RNA preparation

Abstract

The present invention relates to a suitable stored aqueous RNA composition comprising Tris, a sugar and a phosphate anion. The present invention also relates to a method of preparing such an aqueous RNA composition, and to its use in the therapy and prevention of infectious diseases.

Description

Technical Field

[0001] This invention relates to aqueous RNA formulations in which RNA stability is improved. Background Technology

[0002] In recent years, nucleic acids (especially RNA molecules, such as mRNA and siRNA) have seen tremendous development in the application of therapy and other fields, such as crop protection. RNA-based therapeutics include antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), and single-stranded guide RNA (sgRNA) mediated CRISPR-Cas systems.

[0003] RNA molecules are considered much safer than DNA because they can be rapidly cleared from the organism and cannot integrate into the genome to affect gene expression in an uncontrolled manner. RNA therapeutics are also less likely to cause serious side effects, such as autoimmune diseases or the development of anti-DNA antibodies (Bringmann et al., 2010). RNA transfection only requires insertion into the cytoplasm of the cell, which is much easier to achieve than insertion into the cell nucleus.

[0004] Delivery systems, such as lipid nanoparticles (LNPs), are used to protect RNA from degradation and help cells take it up.

[0005] One challenge in formulating RNA into LNPs is maintaining RNA quality over time. Therefore, there is a need in the art for improved RNA formulations. Summary of the Invention

[0006] In one aspect, the present invention provides an aqueous composition comprising an RNA molecule, a delivery system, Tris, and a sugar. Suitably, the aqueous composition further comprises a phosphate anion.

[0007] In another aspect, the present invention provides an aqueous composition for therapeutic purposes, suitably used as a vaccine.

[0008] In another aspect, the present invention provides a method for treating or preventing disease, comprising administering the aqueous composition of the present invention to a patient in need of such treatment.

[0009] In another aspect, the present invention provides a method for treating or preventing infectious diseases, comprising administering the aqueous composition of the present invention to a patient in need of such treatment.

[0010] In another aspect, the present invention provides the use of the aqueous compositions of the present invention in the preparation of pharmaceuticals or vaccines.

[0011] In another aspect, the present invention provides a method for preparing the aqueous composition of the present invention, comprising: a) Provide an initial composition comprising an RNA molecule, preferably mRNA, and a delivery system, preferably LNP. b) Transfer the initial composition to the first buffer system to obtain the intermediate composition. c) Dilute the intermediate composition with a dilution buffer to obtain the aqueous composition of the present invention. Attached Figure Description

[0012] Figure 1 - LNP size (z-mean (nm)) and PdI of formulations 1-6 (labeled as 1-PSN, 2-Na_K_2, 3-Hist, 4-HistArg, 5-TSN, 6-TSG) measured by DLS at T0, after 8 days at -20°C, and after 14 days at +5°C.

[0013] Figure 2 - Formulations 1-6 (labeled as 1-PSN, 2-Na_K_2, 3-Hist, 4-HistArg, 5-TSN, 6-TSG), LEP measured by HPLC at T0, after 8 days at -20°C, after 14 days at +5°C, and after 14 days at +25°C.

[0014] Figure 3 - Formulations 5 (TSN) and 12 (TSG') mRNA integrity after 21 days at +25°C.

[0015] Figure 4 - Formulations 5 (TSN) and 12 (TSG'), LEP after 21 days at +25°C.

[0016] Figure 5 - Encapsulation efficiency (%) of formulations 1 (PSN) and 7-10 (labeled 7-TTS, 8-TTS', 9-TSN, 10-TSN') over 90 days at -20°C, +5°C, and -70°C; over 30 days at +25°C; over 1 month at -20°C followed by 2 months at +5°C; and over 14 days at -70°C followed by 2.5 months at -20°C.

[0017] Figure 6 - LNP size (z-mean (nm) of formulations 1 (PSN) and 7-10 (labeled 7-TTS, 8-TTS', 9-TSN, 10-TSN') over 90 days at -20°C, +5°C, and -70°C; over 30 days at +25°C; over 1 month at -20°C followed by 2 months at +5°C; and over 14 days at -70°C followed by 2.5 months at -20°C.

[0018] Figure 7 - Formulation 1 (PSN) and 7-10 (labeled 7-TTS, 8-TTS', 9-TSN, 10-TSN') within 90 days at -20°C, +5°C, and -70°C; within 30 days at +25°C; within 1 month at -20°C followed by 2 months at +5°C; and within 14 days at -70°C followed by 2.5 months at -20°C.

[0019] Figure 8 - In vitro expression of formulation 1 (PSN) and 7-10 (labeled as 7-TTS, 8-TTS', 9-TS, 10-TS') over 30 days at -20°C, +5°C, and +25°C.

[0020] Figure 9 - LNP size (z-mean (nm) and PdI) of formulations 1 (PSN pH 7.4), 9 (TSN pH 7.4) and 11 (TSN pH 8.0) over 90 days at -20°C, +5°C and +25°C.

[0021] Figure 10 - Encapsulation efficiency (%) of formulations 1 (PSN pH 7.4), 9 (TSN pH 7.4) and 11 (TSN pH 8.0) over 90 days at -20°C, over 50 days at +5°C, and over 50 days at +25°C.

[0022] Figure 11 - mRNA integrity (%) and LEP (%) of formulations 1 (PSN pH 7.4), 9 (TSN pH 7.4) and 11 (TSN pH 8.0) over 50 days at -20°C, +5°C and +25°C.

[0023] Figure 12 - Efficacy (%) of formulations 1 (PSN pH 7.4), 6 (TSG), 9 (TSN 7.4) and 11 (TSN 8) over 30 days at -20°C, +5°C and +25°C.

[0024] Figure 13 - LNP size (z-mean (nm) and PdI) of formulations 6 (TSG), 9 (TSN) and 12 (TSG') over time at -20°C (120 days), +5°C (90 days) and +25°C (90 days).

[0025] Figure 14 - Encapsulation efficiency (%) of formulations 6 (TSG), 9 (TSN) and 12 (TSG') over time at -20°C (120 days), +5°C (90 days) and +25°C (90 days).

[0026] Figure 15 - mRNA integrity (%) and LEP (%) of formulations 6 (TSG), 9 (TSN), and 12 (TSG') over time at -20°C (120 days), +5°C (90 days), and +25°C (90 days).

[0027] Figure 16 - Mice immunized intramuscularly with formulations A (PSN), B (PSN / TSN), and C (PSN / TSG) containing LNP-formulated mRNA encoding influenza antigens. Muscle swelling scores at T0, after 21 days at 25°C, and after 40 days at 40°C (PSN only). The mean score for each group is plotted, with vertical lines representing SD.

[0028] Figure 17 - Mice immunized intramuscularly with formulations A (PSN), B (PSN / TSN), and C (PSN / TSG) containing LNP-formulated mRNA encoding influenza antigens. Release of pro-inflammatory cytokines (IFN-γ; CCL2, CXCL10) at T0, after 21 days at 25°C, and after 40 days at 40°C (PSN only). Each point represents an individual animal, and the lines depict the geometric mean (GM) and 95% confidence interval (CI). LLOQ = lower limit of quantitation. Non-inferiority (NI) between induced responses is depicted if the 90% lower limit of the CI for the geometric mean ratio (GMR) is >0.33. ND = inconclusive, as a 90% lower limit of CI ≤0.33 and non-inferiority cannot be demonstrated.

[0029] Figure 18 - Mice immunized intramuscularly with formulations A (PSN), B (PSN / TSN), and C (PSN / TSG), which contain LNP-formulated mRNA encoding influenza antigens. Humoral response: HI and NI titers at T0, 21 days after administration of the formulations stored at 25°C, and 40 days after administration of the formulations stored at 40°C (PSN only), on day 21 and day 35.

[0030] Figure 19 - Mice immunized intramuscularly with formulations A (PSN), B (PSN / TSN), and C (PSN / TSG), which contain LNP-formulated mRNA encoding influenza antigens. Cellular response: CD4 and CD8 titers at day 21 and day 35 after injection of formulations at T0, after 21 days of storage at 25°C, and after 40 days of storage at 40°C (PSN only). Detailed Implementation

[0031] In one aspect, the present invention provides an aqueous composition comprising an RNA molecule, a delivery system, Tris, and a sugar.

[0032] Suitably, the aqueous composition of the present invention comprises RNA molecules, a delivery system, Tris, sugars, and phosphate anions.

[0033] The aqueous compositions of the present invention contain Tris. As used herein, " Tris "Tris" refers to tris(hydroxymethyl)aminomethane, also known as "tromethamine" or "THAM". Tris is an organic compound with the chemical formula (HOCH2)3CNH2, which can be used as a buffer in drug solutions.

[0034] Suitably, Tris is present at concentrations between 5 mM and 50 mM, and suitably between 7.5 mM and 30 mM, for example at concentrations of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mM. Suitably, Tris is present at a concentration of 15 mM. In another embodiment, Tris is present at a concentration of 30 mM. In another embodiment, Tris is present at a concentration of 10 mM. The aqueous composition of the present invention comprises sugar. A “sugar” (or “carbohydrate”) consists of one or more units of the general formula CnH2nOn. Sugar units exist in a cyclic or short-chain conformation and typically contain five or six carbon atoms. Sugars can be classified as monosaccharides, disaccharides, oligosaccharides, and polysaccharides according to the number of units. A monosaccharide consists of one sugar unit. A disaccharide consists of two identical or different sugar units. Oligosaccharides typically consist of 3 to 20 identical or different sugar units. Polysaccharides typically consist of longer chains of identical or different sugar units.

[0035] Suitably, the sugar is a disaccharide (e.g., sucrose, trehalose). Suitably, the sugar is a disaccharide selected from sucrose and trehalose or a combination thereof. In a particularly suitable embodiment, the sugar is sucrose. In another embodiment, the sugar is a combination of sucrose and trehalose. Suitably, the sugar acts as a cryoprotectant and / or an isotonic regulator. As used herein, " cryoprotectant "A type of excipient that maintains structural integrity (e.g., LNP structural integrity) during freezing. As used herein, " Isotonic adjustment Agent "It is an excipient that can regulate osmotic pressure through colligative properties."

[0036] Suitably, the sugar is present in the aqueous composition at a concentration between 50 mM and 700 mM, preferably between 100 mM and 600 mM, for example at a concentration of about 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM, preferably at a concentration of about 300 mM.

[0037] Suitably, the sugar is sucrose and is present at a concentration between 50 mM and 700 mM, preferably between 100 mM and 600 mM, for example at a concentration of about 150, 200, 250, 300, 350, 400, 450, 500, or 550 mM. More preferably, the sucrose is present at a concentration of about 300 mM.

[0038] In another suitable embodiment, the sugar is a combination of sucrose and trehalose, and the total concentration of sucrose and trehalose is between 50 mM and 700 mM, suitably between 100 mM and 600 mM, for example, about 150, 200, 250, 300, 350, 400, 450, 500, or 550 mM. In one embodiment, both sucrose and trehalose are present at a concentration of about 150 mM. In one embodiment, sucrose is present at a concentration of 50 mM and trehalose is present at a concentration of 550 mM.

[0039] The aqueous composition of the present invention suitably contains phosphate anions (PO4). 3- Typically, phosphate anions associate with cations to form salts, such as NaK2 phosphate or Na / Na2 phosphate.

[0040] Suitablely, the phosphate anion is present at a concentration of 0.5 mM to 15 mM, suitablely 1 mM to 10 mM, more suitablely 1 mM to 5 mM, for example at a concentration selected from about 1, 2, 3, 4 or 5 mM. Suitablely, the phosphate anion is present at a concentration of about 3 mM.

[0041] Suitably, the aqueous composition of the present invention further comprises sodium chloride. In one embodiment, sodium chloride is present at a concentration between 5 mM and 50 mM, preferably between 10 mM and 40 mM, more preferably between 15 mM and 30 mM, for example at a concentration of about 15, 20, 21, 22, 22.5, 23, 24, 25, or 30 mM. Suitably, sodium chloride is present at a concentration of about 22.5 mM.

[0042] Suitably, the pH of the aqueous composition of the present invention is between 6 and 9, preferably between 6.5 and 8.5, for example, about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. Suitably, the pH of the aqueous composition is about 7.4 or about 8.0. More preferably, the pH of the aqueous composition is about 7.4.

[0043] In some embodiments, the osmolar concentration of the aqueous composition of the present invention is less than 450 mOsmol / kg or less than 400 mOsmol / kg.

[0044] In some embodiments, the osmolar concentration of the aqueous composition of the present invention is 100 mOsmol / kg to 400 mOsmol / kg, 150 mOsmol / kg to 400 mOsmol / kg, 200 mOsmol / kg to 400 mOsmol / kg, or 300 mOsmol / kg to 400 mOsmol / kg.

[0045] The aqueous compositions of the present invention may optionally contain glycerol. Suitably, when glycerol is present in the aqueous composition, its concentration is from 10 mM to 200 mM, preferably from 25 mM to 175 mM, for example about 25, 50, 75, 100 or 150 mM, more preferably about 25 mM or about 100 mM.

[0046] In some embodiments, the aqueous composition of the present invention comprises glycerol, and the total concentration of sugar and glycerol is between 50 mM and 700 mM, suitably between 100 mM and 600 mM, for example about 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM, and wherein, for example, the sugar is sucrose and is present at a concentration of about 150 mM, and the glycerol is present at a concentration of about 100 mM.

[0047] In one embodiment of the aqueous composition of the present invention, a. Tris exists at concentrations ranging from 5 mM to 50 mM, preferably from 7.5 mM to 30 mM. b. The sugar, preferably sucrose, is present at a concentration ranging from 50 mM to 700 mM, preferably from 100 mM to 600 mM. c. Phosphate anions are present at concentrations ranging from 0.5 mM to 15 mM, preferably from 1 mM to 5 mM. d. Sodium chloride is present at a concentration ranging from 5 mM to 50 mM, preferably from 15 mM to 30 mM, and The pH of the aqueous composition is between 6 and 9, preferably between 6.5 and 8.5.

[0048] In one embodiment of the aqueous composition of the present invention, a. Tris exists at concentrations ranging from 5 mM to 50 mM, preferably from 7.5 mM to 30 mM. b. The sugar, preferably sucrose, is present at a concentration ranging from 50 mM to 700 mM, preferably from 100 mM to 600 mM. c. Glycerol is present at concentrations ranging from 10 mM to 200 mM, preferably from 25 mM to 175 mM. d. The phosphate anion exists at a concentration ranging from 0.5 mM to 15 mM, preferably from 1 mM to 5 mM. e. Sodium chloride is present at concentrations ranging from 5 mM to 50 mM, suitably from 15 mM to 30 mM, and The pH of the aqueous composition is between 6 and 9, preferably between 6.5 and 8.5.

[0049] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. c. The phosphate anion exists at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 7.4.

[0050] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 30 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. c. The phosphate anion exists at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 7.4.

[0051] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 550 mM. c. The phosphate anion exists at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 7.4.

[0052] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. c. The phosphate anion exists at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 8.0.

[0053] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 30 mM. b. The sugar is sucrose and exists at a concentration of approximately 150 mM. c. The phosphate anion exists at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM. e. Glycerin exists at a concentration of approximately 100 mM, and The pH of this aqueous composition is approximately 7.4.

[0054] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 10 mM. b. The sugar is sucrose and exists at a concentration of approximately 225 mM. c. The phosphate anion exists at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM. e. Glycerin is present at a concentration of approximately 25 mM, and The pH of this aqueous composition is approximately 7.4.

[0055] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is a combination of sucrose at a concentration of approximately 150 mM and trehalose at a concentration of approximately 150 mM. c. The phosphate anion exists at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 7.4.

[0056] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is a combination of sucrose at a concentration of approximately 50 mM and trehalose at a concentration of approximately 500 mM. c. The phosphate anion exists at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 7.4.

[0057] In some embodiments, the aqueous composition of the present invention is substantially free of phosphate anions. In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 7.4.

[0058] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 30 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 7.4.

[0059] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 550 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 7.4.

[0060] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 8.0.

[0061] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 30 mM. b. The sugar is sucrose and exists at a concentration of approximately 150 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM. e. Glycerin exists at a concentration of approximately 100 mM, and The pH of this aqueous composition is approximately 7.4.

[0062] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 10 mM. b. The sugar is sucrose and exists at a concentration of approximately 225 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM. e. Glycerin is present at a concentration of approximately 25 mM, and The pH of this aqueous composition is approximately 7.4.

[0063] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is a combination of sucrose at a concentration of approximately 150 mM and trehalose at a concentration of approximately 150 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 7.4.

[0064] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is a combination of sucrose at a concentration of approximately 50 mM and trehalose at a concentration of approximately 500 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of this aqueous composition is approximately 7.4.

[0065] In some embodiments, the aqueous composition of the present invention is substantially free of sodium chloride.

[0066] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. c. The phosphate anion exists at a concentration of approximately 3 mM, and The pH of this aqueous composition is approximately 7.4.

[0067] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 30 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. c. The phosphate anion exists at a concentration of approximately 3 mM, and The pH of this aqueous composition is approximately 7.4.

[0068] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 550 mM. c. The phosphate anion exists at a concentration of approximately 3 mM, and The pH of this aqueous composition is approximately 7.4.

[0069] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. c. The phosphate anion exists at a concentration of approximately 3 mM, and The pH of this aqueous composition is approximately 8.0.

[0070] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 30 mM. b. The sugar is sucrose and exists at a concentration of approximately 150 mM. c. The phosphate anion exists at a concentration of approximately 3 mM. e. Glycerin exists at a concentration of approximately 100 mM, and The pH of this aqueous composition is approximately 7.4.

[0071] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 10 mM. b. The sugar is sucrose and exists at a concentration of approximately 225 mM. c. The phosphate anion exists at a concentration of approximately 3 mM. e. Glycerin is present at a concentration of approximately 25 mM, and The pH of this aqueous composition is approximately 7.4.

[0072] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is a combination of sucrose at a concentration of approximately 150 mM and trehalose at a concentration of approximately 150 mM. c. The phosphate anion exists at a concentration of approximately 3 mM, and The pH of this aqueous composition is approximately 7.4.

[0073] In one embodiment of the aqueous composition of the present invention, a. Tris exists at a concentration of approximately 15 mM. b. The sugar is a combination of sucrose at a concentration of approximately 50 mM and trehalose at a concentration of approximately 500 mM. c. The phosphate anion exists at a concentration of approximately 3 mM, and The pH of this aqueous composition is approximately 7.4.

[0074] Suitably, the aqueous compositions according to the invention remain stable over time. This stability over time can be assessed by monitoring changes in the quality attributes of the aqueous compositions over time. Physicochemical QA methods that can be used to monitor changes in the quality attributes of the aqueous compositions over time include mRNA-LNP size (z-mean) and polydispersity index (PdI), encapsulation efficiency (EE), mRNA integrity, and late elution peak (LEP) of mRNA, sometimes also referred to as late elution material.

[0075] Suitably, the aqueous composition according to the invention remains stable after the thawing step. Stability after the thawing step can be assessed by monitoring changes in the quality properties of the aqueous composition over time. Physicochemical QA methods that can be used to monitor changes in the quality properties of the aqueous composition over time include mRNA-LNP size (z-mean) and polydispersity index (PdI), encapsulation efficiency (EE), mRNA integrity, and mRNA late elution peak (LEP).

[0076] mRNA-LNP size (z-mean) and Multi-dispersion index (PdI) allows for the characterization and monitoring of LNP particle size distribution during heat stress. In particular, particle size indicates the mean of the particle size distribution, while PdI indicates its dispersion. mRNA / LNP size and polydispersity index (PdI) can be measured by methods well known in the art. For example, dynamic light scattering (DLS) can be used to monitor mRNA-LNP size (Z-mean) and polydispersity index (PdI), as described in Example 1.

[0077] Suitably, in the aqueous composition according to the invention, the z-mean mRNA / LNP size increases or decreases by no more than 10% over a period of 90 days at -20°C, preferably no more than 5%.

[0078] Suitably, in the aqueous composition according to the invention, the z-average mRNA / LNP size is maintained between 60 nm and 100 nm, suitably between 70 nm and 90 nm, and more preferably between 75 nm and 85 nm over a period of 90 days at -20°C.

[0079] Suitably, in the aqueous composition according to the invention, the z-mean mRNA / LNP size increases or decreases by no more than 10% over a period of 90 days at +5°C, preferably no more than 5%.

[0080] Suitably, in the aqueous composition according to the invention, the z-mean mRNA / LNP size is maintained between 60 nm and 100 nm, preferably between 70 nm and 90 nm, and more preferably between 75 nm and 85 nm over a period of 90 days at +5°C.

[0081] Suitably, in the aqueous composition according to the invention, the z-mean mRNA / LNP size increases or decreases by no more than 10%, preferably no more than 5%, over a period of 30 days, preferably 60 days, and more preferably 90 days at +25°C.

[0082] Suitably, in the aqueous composition according to the invention, the z-mean mRNA / LNP size is maintained between 60 nm and 100 nm, suitably between 70 nm and 90 nm, and more preferably between 75 nm and 85 nm for 30 days, suitably between 60 days, and more preferably between +25°C and 90 days.

[0083] Suitably, in the aqueous composition according to the invention, the z-mean mRNA / LNP size increases or decreases by no more than 10% over a period of 90 days at -20°C and over a period of 90 days at +5°C or +25°C, preferably no more than 5%.

[0084] Suitably, in the aqueous composition according to the invention, the z-mean mRNA / LNP size is maintained between 60 nm and 100 nm, suitably between 70 nm and 90 nm, and more preferably between 75 nm and 85 nm, over a period of 90 days at -20°C and 90 days at +5°C or +25°C.

[0085] Encapsulation efficiency Encapsulation efficiency (EE) is the percentage of mRNA encapsulated in LNP relative to the total mRNA concentration in the composition. Encapsulation efficiency is believed to be closely related to mRNA activity; reduced encapsulation may lead to a decrease in the amount of mRNA delivered to cells, resulting in decreased protein expression efficiency. Encapsulation efficiency can be measured using methods well known in the art. For example, the Ribogreen assay can be used to monitor encapsulation efficiency (EE), as described in Example 1.

[0086] Suitably, in the aqueous composition according to the invention, the encapsulation efficiency is greater than 75% over a period of 90 days at -20°C, preferably greater than 80%, and more preferably greater than 85%.

[0087] Suitably, in the aqueous composition according to the invention, the encapsulation efficiency is greater than 75% over a period of 90 days at +5°C, preferably greater than 80%, and more preferably greater than 85%.

[0088] Suitably, in the aqueous composition according to the invention, the encapsulation efficiency is greater than 75% over a period of 90 days at +25°C, preferably greater than 80%, and more preferably greater than 85%.

[0089] Suitably, in the aqueous composition according to the invention, the encapsulation efficiency is greater than 75% over a period of 90 days at -70°C, preferably greater than 80%, and more preferably greater than 85%.

[0090] Suitably, in the aqueous composition according to the invention, the encapsulation efficiency is greater than 75% over a period of 90 days at -20°C and over a period of 90 days at +5°C or +25°C, preferably greater than 80%, and more preferably greater than 85%.

[0091] mRNA integrity The relative amount (%) of intact mRNA molecules relative to fragments that may result from mRNA degradation is measured. mRNA integrity can be monitored to detect mRNA degradation that may occur over time due to the unstable nature of the molecules. mRNA integrity is believed to directly affect mRNA efficacy, as reduced integrity may lead to decreased protein expression levels. Additionally, mRNA fragments may lead to increased reactivity. mRNA integrity can be measured using methods well-known in the art. For example, reversed-phase ion-pair high-performance liquid chromatography (RP-IP HPLC) can be used to monitor mRNA integrity, as described in Example 1. Capillary electrophoresis (CGE) is another suitable method for evaluating mRNA integrity.

[0092] Suitably, in the aqueous composition according to the invention, the mRNA integrity is greater than 70%, preferably greater than 75%, over a period of 90 days at -20°C.

[0093] Suitably, in the aqueous composition according to the invention, the mRNA integrity is greater than 70%, preferably greater than 75%, over a period of 90 days at +5°C.

[0094] Suitably, in the aqueous composition according to the invention, the mRNA integrity is greater than 50%, preferably greater than 55%, over a period of 30 days at +25°C.

[0095] Suitably, in the aqueous composition according to the invention, mRNA integrity is greater than 70% over a period of 90 days at -20°C and over a period of 90 days at +5°C or +25°C, preferably greater than 75%.

[0096] Late Elution Peak (LEP) of mRNA This corresponds to lipid mRNA adducts derived from electrophilic impurities of ionizable cationic lipid components through covalent addition with mRNA nucleobases. This phenomenon is believed to render mRNA untranslatable, leading to loss of protein expression (M Packer et al., Nature Communications, 2021). The relative amount (%) of mRNA LEP can be measured using methods well-known in the art. For example, RP-HPLC can be used to monitor mRNA LEP.

[0097] Suitably, in the aqueous composition according to the invention, the relative amount of mRNA LEP is less than 2% over a period of 90 days at -20°C, preferably less than 1.5%, and more preferably less than 1%.

[0098] Suitably, in the aqueous composition according to the invention, the relative amount of mRNA LEP is less than 2% over a period of 90 days at +5°C, suitably less than 1.5%, and more preferably less than 1%.

[0099] Suitably, in the aqueous composition according to the invention, the relative amount of mRNA LEP is less than 2% over a period of 30 days at +25°C, preferably less than 1.5%, and more preferably less than 1%.

[0100] efficacy Efficacy can be considered as the expression of the encoded peptides or proteins after the aqueous composition is administered to cells. If the aqueous composition is a vaccine, efficacy can be considered as the induction of specific antibody titers after the aqueous composition is administered to cells, and / or the induction of neutralizing antibody titers after the aqueous composition is administered to cells, and / or the induction of antigen-specific T cell responses after the aqueous composition is administered to cells. Efficacy is typically determined using cell-based protein expression assays.

[0101] Suitably, the efficacy of the aqueous composition of the present invention decreases by less than 30%, preferably less than 20%, and more preferably less than 10% over a period of 90 days at -20°C.

[0102] Suitably, the efficacy of the aqueous composition of the present invention decreases by less than 30%, preferably less than 20%, and more preferably less than 10% over a period of 90 days at +5°C.

[0103] Suitably, the efficacy of the aqueous composition of the present invention decreases by less than 30%, preferably less than 20%, and more preferably less than 10% over a period of 90 days at +25°C.

[0104] Suitably, in the aqueous composition according to the invention, after a thawing step from, for example, -20°C to 5°C, the encapsulation efficiency is greater than 75%, preferably greater than 80%, and more preferably greater than 85%.

[0105] Suitably, in the aqueous composition according to the invention, after a thawing step from, for example, -20°C to 5°C, the z-average mRNA / LNP size increases or decreases by no more than 10%, suitably no more than 5%.

[0106] Suitably, the potency of the aqueous composition according to the invention decreases by less than 30%, preferably less than 20%, and more preferably less than 10% after a thawing step from, for example, -20°C to 5°C.

[0107] The aqueous compositions of the present invention comprise a delivery system for RNA. A series of delivery systems have been described that can be used to encapsulate RNA (or form complexes with it) to protect the RNA and facilitate its delivery to target cells. In a preferred embodiment, the delivery system is lipid nanoparticles (LNPs).

[0108] the term" lipid nanoparticles "(or "LNP")" refers to non-viral particles capable of encapsulating nucleic acid molecules (such as RNA). LNPs are not limited to any particular form and include any form produced when ionizable (or cationic) lipids, and optionally one or more other lipids, are combined, for example, in an aqueous environment and / or in the presence of nucleic acids (such as RNA). For example, liposomes, lipid complexes, lipoplexes, etc., fall within the scope of lipid nanoparticles (LNPs). LNP delivery systems and methods for their preparation are known in the art. Suitably, at least about 80%, 85%, 90%, and 95% of LNPs have a spherical morphology. LNPs are generally suitable for intramuscular, intradermal, and / or intravenous administration.

[0109] Suitably, the LNP comprises (i) ionizable amino lipids, (ii) neutral lipids, suitably DSPC, (iii) sterols, suitably cholesterol; and (iv) PEGylated lipids.

[0110] In this article, " Ionizable amino lipids "It is an ionizable lipid containing an amino group." Ionizable lipids "Ionizable amino lipids are lipids that undergo protonation when the pH drops below the pKa of the ionizable group, but gradually become more neutral at higher pH values. At pH values ​​below pK, these lipids can associate with negatively charged nucleic acids. In some embodiments, ionizable amino lipids comprise zwitterionic lipids that are positively charged when the pH decreases. Suitably, the ionizable amino lipids are selected from tertiary amine ionizable amino lipids and secondary amine ionizable amino lipids. More preferably, the ionizable amino lipids are tertiary amine ionizable amino lipids."

[0111] In some embodiments, ionizable amino lipids, as defined herein, have at least one protonable or deprotonable group, such that the lipid is positively charged at a pH equal to or below physiological pH (e.g., pH 7.4), and neutral at a second pH, suitably equal to or above physiological pH. It should be understood that the addition or removal of protons with pH changes is an equilibrium process, and the reference to charged or neutral lipids refers to the properties of the dominant substance and does not require all lipids to be present in a charged or neutral form. Lipids having more than one protonable or deprotonable group, or being zwitterionic, are not excluded and may also be suitable in the context of this invention. In some embodiments, the pKa of the protonable group of the protonable lipid is in the range of about 4 to about 11, for example, about 5 to about 7.

[0112] Ionizable amino lipids can be cationic. Unless the specific context clearly distinguishes them, the term "ionizable amino lipid" should be used with caution. Cations "" indicates that the corresponding structure is positively charged, either permanent or non-permanent but responsive to certain conditions (such as pH). Therefore, the term "cationic" encompasses " Permanent cations "and" cationic "Both. Term" Permanent Cations This indicates that, for example, the corresponding compound, group, or atom carries a positive charge at any pH or hydrogen ion activity in its environment. Typically, the positive charge comes from the presence of quaternary nitrogen atoms.

[0113] As used in this article, the term " cationic "" indicates that the compound, group, or atom is positively charged at lower pH levels in its environment and uncharged at higher pH levels. Additionally, in non-aqueous environments where the pH cannot be determined, cationizable compounds, groups, or atoms are positively charged at high hydrogen ion concentrations and uncharged at low hydrogen ion concentrations or activities. The pH or hydrogen ion concentration at which a cationizable or polycationizable compound is charged or uncharged depends on the individual characteristics of the compound, particularly the pKa of the corresponding cationizable group. In diluted aqueous environments, the proportion of positively charged cationizable compounds, groups, or atoms can be estimated using the so-called Henderson-Hasselbalch equation, well known to those skilled in the art. In some embodiments, it is suitable that the cationizable compound or portion is primarily neutral at physiological pH levels (e.g., about 7.0–7.4) but becomes positively charged at lower pH levels. In some embodiments, the pKa of the cationizable compound or portion ranges from about 5 to about 7.

[0114] Suitable ionizable amino lipids include, but are not limited to, N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), ckk-E12, ckk, 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linoleoyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5, 1,2-dilinoleoylcarbamoyloxy-3-dimethylaminopropane (Dlin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (Dlin-DAC), 1,2-dilinoleoyloxy-3-morpholinylpropane (Dlin-MA), 1,2-dilinoleoyl-3- Dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (Dlin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (Dlin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (Dlin-TMA.Cl), HGT5000, HGT5001, DMDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCDAP, Klin-K-DMA, Dlin-K-XTC2-DMA, XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), HGT4003, 1,2-dilinoleyl-3-trimethylaminopropane chloride (Dlin-TAP).Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazinyl)propane (Dlin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DlinAP), 3-(N,N-dilinoleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (Dlin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (Dlin-K-DMA) or analogues thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9 ,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxacyclopenta-5-amine, (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (MC3), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxacyclopenta-5-amine), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperyl) (1-azine-1-yl)ethylazinediyl)bisdodecane-2-ol (C12-200), 2,2-dilinyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (Dlin-K-C2-DMA), 2,2-dilinyl-4-dimethylaminomethyl-[1,3]-dioxolane (Dlin-K-DMA), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N,N,16-bisundecyl-4,7,10,13-tetraazahexahexadecane-1,16-diamide), (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraene-19 3-(dimethylamino)butyrate (Dlin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptadecano-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropyl-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptadecano-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbut-1-amine (MC4 ether), LIPOFECTIN® (commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3-phosphatidylethanolamine (DOPE), sourced from GIBCO / BRL, Grand Island, NY)); dilinoleylmethyl-4-dimethylaminobutyrate (Dlin-MC3-DMA); and TRANSFECTAM® (a commercially available cationic lipid comprising bis(octadecylamidoglycylcarboxylic)spermine (DOGS) in ethanol, from Promega Corp., Madison, Wis.), or any combination of the foregoing substances. Other suitable ionizable amino lipids for use in the aqueous compositions and methods of the present invention include those described in International Patent Publications WO2010053572 (and in particular, CI 2-200 as described in paragraph

[00225] ) and WO2012170930 (both incorporated herein by reference), as well as HGT4003, HGT5000, HGT5001, HGT5002 (see US20150140070A1).

[0115] In some embodiments, the ionizable amino lipid is an ionizable amino alcohol lipid.

[0116] Ionizable amino alcohol lipids can be prepared by the method described in U.S. Patent No. 8,450,298 (which is incorporated herein by reference in its entirety).

[0117] Suitable ionizable amino lipids may also be compounds disclosed in Tables 1, 2 and 3 of WO2017075531A1 (which is incorporated herein by reference) and as defined in claims 1-24.

[0118] In another embodiment, suitable ionizable amino lipids may also be compounds disclosed in WO2015074085A1 (i.e., ATX-001 to ATX-032 or the compounds specified in claims 1-26), U.S. Applications 61 / 905,724 and 15 / 614,499, or U.S. Patents 9,593,077 and 9,567,296 (all of which are incorporated herein by reference in their entirety).

[0119] In other embodiments, suitable ionizable amino lipids may also be compounds disclosed in WO2017117530A1 (which is incorporated herein by reference in its entirety) (i.e. lipids 13, 14, 15, 16, 17, 18, 19, 20, or the compounds specified in the claims).

[0120] In some embodiments, the ionizable amino lipids may also be selected from the lipids disclosed in WO2018078053A1 (i.e., lipids of formulas I, II, and III of WO2018078053A1 or lipids as specified in claims 1 to 12 of WO2018078053A1), the disclosure of WO2018078053A1 being incorporated herein by reference in its entirety. In this context, the lipids disclosed in Table 7 of WO2018078053A1 (e.g., lipids derived from formulas I-1 to I-41) and the lipids disclosed in Table 8 of WO2018078053A1 (e.g., lipids derived from formulas II-1 to II-36) may be suitably used in the context of this invention. Therefore, formulas I-1 to I-41 and formulas II-1 to II-36 of WO2018078053A1, and the specific disclosures associated therewith, are hereby incorporated herein by reference.

[0121] In some embodiments, the ionizable amino lipid may be derived from Formula III of the published PCT patent application WO2018078053A1. Therefore, Formula III of WO2018078053A1 and the specific disclosure therein are hereby incorporated by reference.

[0122] In some embodiments, the ionizable amino lipids are selected from structures III-1 to III-36 of Table 9 of the published PCT patent application WO2018078053A1. Therefore, formulas III-1 to III-36 of WO2018078053A1 and the specific disclosures thereto are hereby incorporated by reference.

[0123] In some embodiments, the ionizable (cationic) lipid has Formula III: (III) Or its pharmaceutically acceptable salts, tautomers or stereoisomers, wherein: L 1 or L 2 Each is independently -O(C=O)- or –(C=O)O-; G 1 and G 2 Each is an unsubstituted C1-C. 12 Alkylene or C1-C 12 alkenyl; G 3 It is C1-C 24 Alkylene, C1-C 24 alkenyl, C3-C8 cycloalkylene or C3-C8 cycloalkenyl; R 1 and R 2Each is independently either a branched chain or a straight chain C6-C 24 Alkyl or C6-C 24 alkenyl; R 3 Is it H or OR? 5 CN, -C(=O)OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 It is C1-C 12 alkyl; R 5 It is H or C1-C6 alkyl.

[0124] In some embodiments, the ionizable (cationic) lipid has Formula III: (III) Or its pharmaceutically acceptable salts, tautomers or stereoisomers, wherein: L 1 or L 2 Each is independently -O(C=O)- or –(C=O)O-; G 1 and G 2 Each is an unsubstituted C1-C. 12 Alkylene; G 3 It is C1-C 24 Alkylene; R 1 and R 2 Each is independently either a branched chain or a straight chain C6-C 24 alkyl; R 3 Is it OR 5 ;and R 5 It is H.

[0125] In some embodiments, the ionizable (cationic) lipid has formula III, and wherein R 1 R 2 Or R 1 and R 2 Both have one of the following structures: .

[0126] In some implementations, R 2 It has a structure: .

[0127] In some embodiments, the ionizable (cationic) lipid has the following formula: . In some embodiments, the ionizable (cationic) lipid has the following formula: ; ; ;or .

[0128] In some embodiments, the ionizable (cationic) lipid has formula III-3: (III-3)

[0129] The chemistry term for the lipid of formula III-3, as suitably used herein, is ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), also known as ALC-0315, CAS number 2036272-55-4.

[0130] In some embodiments, the ionizable (cationic) lipid has the following structure: .

[0131] Other suitable ionizable amino acid lipids are disclosed in WO2009086558, WO2009127060, WO2010048536, WO2010054406, WO2010088537, WO2010129709, WO2011153493, WO2013063468, US20110256175, US20120128760, and US2 0120027803, US8158601, WO2016118724, WO2016118725, WO2017070613, WO2017070620, WO2 017099823, WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, W O2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, WO2013086373, WO2013086354, US Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8 ,466,122 and 8,569,256 and U.S. Patent Publications US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, US20130225836, US20140039032 and WO2017112865.Against this backdrop, WO2009086558, WO2009127060, WO2010048536, WO2010054406, WO2010088537, WO2010129709, WO2011153493, WO 2013063468, US20110256175, US20120128760, US20120027803, US8158601, WO2016118724, WO2016118725, WO2017070613, WO2017070620, WO2017099823, WO2012 040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO201102 2460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO20102186 5. The disclosures in WO2008103276, WO2013086373, WO2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333, 8,466,122 and 8,569,256, and U.S. Patent Publications US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, US20130225836 and US20140039032, and WO2017112865 that are specifically relevant to ionizable (cationic) lipids suitable for LNPs are hereby incorporated herein by reference.

[0132] In other embodiments, the ionizable amino lipid is a tertiary amine ionizable amino lipid, suitably selected from... ; ; ; ; ; ; ; ; ;and .

[0133] The amount of ionizable aminolipids can be chosen taking into account the amount of RNA (N / P ratio). In this context, " N / P ratio The N / P ratio is defined as the molar ratio of nitrogen atoms (“N”) in the basic nitrogen-containing group of an ionizable amino lipid to phosphate groups (“P”) in RNA. The N / P ratio can be calculated based on the following: for example, 1 µg of RNA typically contains approximately 3 nmol of phosphate residues, provided that the RNA exhibits a statistical distribution of bases. The N value of an ionizable amino lipid can be calculated based on its molecular weight and the relative abundance of permanent cations and / or cationizable groups. If more than one ionizable amino lipid is present, the N value should be calculated based on all ionizable amino lipids contained in the lipid nanoparticle.

[0134] In one embodiment, the N / P ratio of the aqueous composition is from about 0.1 to about 20. In one embodiment, the N / P ratio of the aqueous composition is from about 1 to about 20, preferably from about 2 to about 15, more preferably from about 3 to about 10, even more preferably from about 4 to about 9, and most preferably from about 6. In one embodiment, the N / P ratio of the aqueous composition is from about 5 to about 20, more preferably from about 10 to about 18, even more preferably from about 12 to about 16, and most preferably from about 14.

[0135] In some embodiments, ionizable amino lipids are present in the LNP in an amount of about 20 mol% to about 70 mol% (based on 100% total lipid moles in the LNP). Suitably, ionizable amino lipids are present in the LNP in an amount of about 30 mol% to about 65 mol%. In one embodiment, ionizable amino lipids are present in the LNP in an amount of about 40 mol% to about 60 mol%, such as about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 mol%. In one embodiment, the ionizable amino lipid is present in the LNP in an amount of about 47 mol% to about 48 mol%, such as about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, and 50.0 mol%, respectively, of which 47.4 mol% is particularly suitable.

[0136] LNPs may contain two or more (different) ionizable amino lipids as defined herein. Ionizable amino lipids can be selected to contribute different advantageous properties. For example, ionizable amino lipids differing in properties such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity can be used in LNPs (or liposomes, nanoliposomes, lipoplexes). In particular, ionizable amino lipids can be selected such that the properties of a mixed LNP are more desirable than those of a single LNP of individual lipids. If more than one type of ionizable amino lipid is present, the above percentages apply to the total amount of ionizable amino lipids.

[0137] In addition to ionizable amino lipids, LNPs typically contain one or more other lipids selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids (such as PEGylated lipids), which together with ionizable amino lipids form LNPs.

[0138] Suitablely, LNP comprises polymer-conjugated lipids. The term "..." Polymer-conjugated lipids "" refers to a molecule that comprises both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is an EG-conjugated lipid. The term "" PEGylated lipids "(or "PEG-modified lipids" or "PEG lipids") refers to a molecule that comprises both a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), etc.

[0139] Polymer-conjugated lipids (such as PEGylated lipids) as defined herein can be used as lipids to reduce aggregation.

[0140] Suitably, the LNP comprises a PEGylated lipid. Suitable PEGylated lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. Representative PEGylated lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the PEGylated lipid is N-[(methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). In some embodiments, the PEGylated lipid is PEG-2000-DMG. In one embodiment, the PEGylated lipid is PEG-c-DOMG. In other embodiments, the LNP comprises PEGylated diacylglycerols (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEGylated succinate diacylglycerols (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), PEGylated ceramides (PEG-cer), or PEGylated dialkoxypropyl carbamates such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0141] In some embodiments, the PEGylated lipids comprise PEG-DMG or PEG-cDMA.

[0142] In embodiments, the PEGylated lipids are suitably derived from formula (IV) of the published PCT patent application WO2018078053A1. Therefore, the PEGylated lipids derived from formula (IV) of the published PCT patent application WO2018078053A1, and the corresponding disclosure thereto, are hereby incorporated by reference.

[0143] In some embodiments, the PEGylated lipids have formula IV: (IV) Where R 8 and R 9 Each is independently a straight or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; And the average value of w ranges from 30 to 60.

[0144] In one implementation, R 8 and R 9 It is a saturated alkyl chain.

[0145] In some embodiments, RNA (suitably mRNA) forms a complex with one or more lipids to form an LNP, wherein the LNP comprises a polymer-conjugated lipid, suitably PEGylated lipid, wherein the PEGylated lipid is suitably derived from formula (Iva) of published PCT patent application WO2018078053A1. Therefore, the PEGylated lipid derived from formula (Iva) of published PCT patent application WO2018078053A1 and the corresponding disclosure thereto are hereby incorporated by reference.

[0146] In some embodiments, the PEGylated lipids have the formula (Iva): (Iva) The average value of n ranges from 30 to 60, such as about 30±2, 32±2, 34±2, 36±2, 38±2, 40±2, 42±2, 44±2, 46±2, 48±2, 50±2, 52±2, 54±2, 56±2, 58±2, or 60±2. In one embodiment, n is about 49. In another embodiment, n is about 45. In other embodiments, the PEGylated lipid has the formula (Iva), where n is selected such that the average molecular weight of the PEGylated lipid is about 2000 g / mol to about 3000 g / mol or about 2300 g / mol to about 2700 g / mol, suitably about 2500 g / mol.

[0147] In some embodiments, the PEGylated lipids have the formula Iva: (Iva) The average value of n ranges from 30 to 60, preferably with an average value of approximately 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, and most preferably with an average value of 49 or 45; or Where n is an integer chosen to make the average molecular weight of the PEGylated lipid approximately 2500 g / mol.

[0148] The chemistry term for the lipid of formula Iva, as suitably used herein, is 2[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide, also known as ALC-0159.

[0149] Other examples of suitable PEGylated lipids in this context are provided in US20150376115A1 and WO2015199952, which are each incorporated herein by reference in their entirety.

[0150] In some implementations, the LNP contains less than about 3, 2, or 1 molar percentage of PEG or PEGylated lipids, based on the total number of moles of lipids in the LNP.

[0151] In other embodiments, the LNP contains about 0.1% to about 20% PEGylated lipids on a molar basis, for example, about 0.5% to about 15%, about 0.5% to about 10%, about 0.5% to about 5%, about 10%, about 5%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.5%, or about 0.3%. In embodiments, based on molar percentages (based on 100% total lipid moles in the LNP), the LNP contains about 1.0% to about 2.0% PEGylated lipids, for example, about 1.2% to about 1.9%, about 1.2% to about 1.8%, about 1.3% to about 1.8%, about 1.4% to about 1.8%, about 1.5% to about 1.8%, about 1.6% to about 1.8%, particularly about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, and most preferably 1.7%. In various embodiments, the molar ratio of ionizable amino lipids to PEGylated lipids ranges from about 100:1 to about 25:1.

[0152] In some embodiments, the LNP contains PEGylated lipids in amounts of about 0.5 mol% to 10 mol%, optionally 0.5 mol% to 5 mol%, or 0.5 mol% to 3 mol%.

[0153] Suitablely, the LNP contains one or more stabilizing lipids (or auxiliary lipids) that stabilize particle formation during particle formulation or manufacturing (e.g., neutral lipids and / or one or more steroids or steroid analogs).

[0154] Suitable stabilizing lipids (or co-lipids) include neutral lipids. The term " neutral lipids "Neutral lipids" refers to any of a variety of lipid substances that exist as uncharged or neutral zwitterions at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0155] In some embodiments, the LNP comprises one or more neutral lipids selected from the group consisting of: distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4- (N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-ditransoleoyl-sn-glycerol-3-phosphatidylethanolamine (transDOPE), sphingomyelin (SM), or mixtures thereof.

[0156] In various embodiments, the molar ratio of ionizable amino lipids to neutral lipids ranges from about 2:1 to about 8:1.

[0157] Suitably, the neutral lipid is 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC). Suitably, the molar ratio of ionizable amino lipids to DSPC can range from about 2:1 to about 8:1.

[0158] Suitable stabilizing lipids (or co-lipids) also include steroids or steroid analogues.

[0159] In some implementations, the steroid is a sterol, preferably cholesterol.

[0160] Suitably, the molar ratio of ionizable amino lipids to cholesterol can range from about 2:1 to about 1:1. In some embodiments, the cholesterol may be PEGylated.

[0161] Sterols may be from about 10 mol% to about 60 mol% of the lipid particles, or from about 25 mol% to about 55 mol%, or from about 25 mol% to about 40 mol%. In one embodiment, sterols are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 mol% of the total lipids present in the lipid particles. In another embodiment, the LNP contains about 5% to about 50% sterols on a molar basis, for example, about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5%, or about 31% on a molar basis (based on the total number of moles of lipids in the lipid nanoparticles).

[0162] Preferably, the LNP comprises ionizable amino acids, PEGylated lipids, neutral lipids, and sterols. Preferably, the LNP comprises ionizable amino acids, PEGylated lipids, neutral lipids, and sterols. More preferably, the LNP comprises ionizable amino acids, PEGylated lipids, neutral lipids, and sterols.

[0163] In one implementation, LNP includes (i) at least one ionizable amino lipid as defined herein, suitably of formula III-3; (ii) at least one neutral lipid as defined herein, suitably 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC). (iii) at least one sterol as defined herein, suitably cholesterol; and (iv) At least one PEGylated lipid as defined herein, such as PEG-DMG or PEG-cDMA, suitably of formula Iva or derived from formula Iva.

[0164] In one embodiment, the ionizable amino lipid has formula III-3, the neutral lipid is DSPC, the steroid is cholesterol, and the PEGylated lipid has formula Iva.

[0165] Suitably, lipids (i) to (iv) form LNPs and are present in a molar ratio of about 20%-60% ionizable amino lipids: 5%-25% neutral lipids: 25%-55% sterols: 0.5%-15% PEGylated lipids. In one embodiment, lipids (i) to (iv) are present in a molar ratio of about 50:10:38.5:1.5. In one embodiment, lipids (i) to (iv) are present in a molar ratio of about 47.5:10.0:40.8:1.7. In one embodiment, lipids (i) to (iv) are present in a molar ratio of about 47.4:10.0:40.9:1.7.

[0166] In one embodiment, LNP contains an ionizable amino lipid of formula III-3, DSPC, cholesterol, and a PEGylated lipid of formula Iva in a molar ratio of approximately 47.4:10.0:40.9:1.7.

[0167] Other useful LNP compositions are described in the following references: WO2012 / 006376; ​​WO2012 / 030901; WO2012 / 031046; WO2012 / 031043; WO2012 / 006378; WO2011 / 076807; WO2013 / 033563; WO2013 / 006825; WO2014 / 136086; WO2015 / 095340; WO2015 / 095346; WO2016 / 037053; WO2017 / 070620, which are incorporated herein by reference.

[0168] Suitably, the average diameter of the LNP is about 50 nm to about 200 nm, about 60 nm to about 200 nm, about 70 nm to about 200 nm, about 80 nm to about 200 nm, about 90 nm to about 200 nm, about 90 nm to about 190 nm, about 90 nm to about 180 nm, about 90 nm to about 170 nm, about 90 nm to about 160 nm, about 90 nm to about 150 nm, about 90 nm to about 140 nm, about 90 nm to about 130 nm, about 90 nm to about 120 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm. The wavelengths are 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm, and are substantially non-toxic. As used herein, the average diameter can be represented by the z-average size determined by dynamic light scattering as is generally known in the art.

[0169] Suitablely, the polydispersity index (PdI) of the LNP is 0.4 or lower, preferably 0.3 or lower. Typically, PdI is determined by dynamic light scattering.

[0170] Suitablely, at least 50%, more preferably at least 60%, 70%, 80%, 85%, 90%, or 95% of the RNA is encapsulated in LNPs. In this context, " Encapsulated RNA "This is understood as RNA (suitably mRNA) that forms a complex with the lipids that form the LNP and / or is contained within the LNP's internal space. The proportion of encapsulated RNA can usually be determined using the RiboGreen assay."

[0171] Suitably, the aqueous composition contains less than about 30%, preferably less than 20%, 15%, 10%, or 5% of unencapsulated RNA (or free RNA). In this context, the term "..." Free RNA "or" Unencapsulated RNA "This is understood as RNA not encapsulated in an LNP as defined herein (suitably mRNA). In therapeutic compositions, free RNA may represent contaminants or impurities."

[0172] The aqueous composition of the present invention contains RNA molecules.

[0173] the term" RNA "RNA" is the common abbreviation for ribonucleic acid. It is a nucleic acid molecule, a polymer composed of nucleotide monomers. These nucleotides are typically monomers of adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP), and cytidine monophosphate (CMP), or their analogues, which are linked together along a so-called backbone. The backbone is usually formed by phosphodiester bonds between the sugar (ribose) of the first monomer and the phosphate moiety of the second adjacent monomer. The specific sequence of monomers, that is, the order in which the bases are linked to the sugar / phosphate backbone, is called the RNA sequence.

[0174] Suitable RNA molecules are selected from antisense RNAs (such as antisense oligonucleotides (ASOs)), small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs), and RNAs that form part of a single-stranded guide RNA (sgRNA)-mediated CRISPR-Cas system.

[0175] Suitably, the amount of RNA (suitably mRNA) in the aqueous composition according to the invention ranges from about 0.1 µg to about 1000 µg per dose, for example from about 1 µg to about 500 µg, particularly from about 2 µg to about 250 µg, for example 2, 3, 4, 5, 10, 15, 20, 25, 30 or 50 µg per dose.

[0176] As used in this article, " dose "" refers to the amount of aqueous composition to be applied to a subject, suitable for human subjects.

[0177] In one embodiment, the volume of the dose is 0.1 ml to 1 ml, suitably 0.2 ml to 0.8 ml, and more preferably 0.3 ml to 0.6 ml. Typically, the volume of the dose is selected from 0.3, 0.4, and 0.5 ml. In one embodiment, the volume of the dose is 0.3 ml. In one embodiment, the volume of the dose is 0.5 ml.

[0178] In this embodiment, the therapeutic composition is a ready-to-use composition. The ready-to-use composition according to the invention is configured for direct administration to a subject and requires no processing / preparation / dilution steps (e.g., for multi-dose compositions) and / or isotonic adjustment (e.g., by dilution with a NaCl solution, etc.).

[0179] Suitably, the amount of RNA in the therapeutic composition is about 100 ng to about 500 µg, 1 µg to about 200 µg, 5 µg to about 100 µg, or 5 µg to about 50 µg. Therefore, the therapeutic composition may contain a dose of RNA of about 100 ng to about 500 µg, 1 µg to about 200 µg, 5 µg to about 100 µg, or 5 µg to about 50 µg.

[0180] In embodiments under this background, the amount of lipids in the therapeutic composition is about 2500 ng to about 12.5 mg, 25 µg to about 5 mg, 125 µg to about 2.5 mg, or 250 µg to about 1.25 mg.

[0181] In embodiments, the total volume of the therapeutic composition is about 50 µl to 2 ml, about 100 µl to 1 ml, about 250 µl to 1 ml, preferably about 500 µl.

[0182] In embodiments under this background, the total volume of the therapeutic composition is about 50 µl to about 2 ml, 100 µl to 1 ml, 250 µl to 1 ml, preferably about 500 µl, and contains a dose of RNA of about 100 ng to about 500 µg, 1 µg to about 200 µg, 5 µg to about 100 µg, or 5 µg to about 50 µg.

[0183] Suitably, the RNA molecule is at least 200 nucleotides long, more preferably at least 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides long. In a preferred embodiment, the RNA molecule is 1000 to 3000 nucleotides long, preferably 1500 to 2000 nucleotides long.

[0184] In a preferred embodiment, the RNA molecule is messenger RNA (mRNA).

[0185] mRNA is a single-stranded RNA molecule corresponding to a gene's genetic sequence, read by ribosomes during protein production. mRNA can be selected from non-replicating mRNA and self-replicating mRNA (or self-amplifying mRNA or SAM). Non-replicating mRNA typically encodes the protein of interest and contains a 5' and / or 3' untranslated region (UTR), a 5' cap, and a poly(A) tail. Self-amplifying mRNA also encodes viral replication mechanisms, enabling intracellular mRNA amplification.

[0186] In one implementation, the RNA molecule is non-replicating mRNA.

[0187] In one implementation, the RNA molecule is a self-replicating mRNA.

[0188] RNA (suitably mRNA) is suitably provided in a purified or substantially purified form, i.e., substantially free of proteins (e.g., enzymes), other nucleic acids (e.g., DNA and nucleoside phosphate monomers), etc., and is typically at least about 50% pure (by weight), and typically at least 90% pure, such as at least 95% or at least 98% pure.

[0189] RNA (suitably mRNA) can be prepared in a variety of ways, such as through complete or partial chemical synthesis, by digesting long nucleic acids using nucleases (e.g., restriction endonucleases), by ligating shorter nucleic acids or nucleotides (e.g., using ligases or polymerases), or from genomic or cDNA libraries. In particular, mRNA can be prepared enzymatically using a DNA template.

[0190] RNA (suitable mRNA) can be artificial nucleic acid.

[0191] As used in this article, the term " Artificial nucleic acid "Artificial nucleic acid" is intended to refer to nucleic acids that are not naturally occurring. In other words, artificial nucleic acids can be understood as non-natural nucleic acid molecules. Such nucleic acid molecules may be non-natural due to their individual sequences (e.g., coding sequences modified with G / C content, UTRs) and / or due to other modifications (e.g., structural modifications of nucleotides). Typically, artificial nucleic acids can be genetically engineered and / or produced to correspond to artificial sequences of desired nucleotides. In this context, an artificial nucleic acid is a sequence that may not be naturally occurring, i.e., a sequence that differs from the wild-type or reference / naturally occurring sequence by at least one nucleotide (through, for example, codon modifications as further specified below). The term "artificial nucleic acid" is not limited to meaning "a single molecule" but is understood to refer to a collection of substantially identical nucleic acid molecules. Therefore, it can refer to multiple substantially identical nucleic acid molecules.

[0192] In some implementations, the mRNA may be a modified and / or stabilized mRNA, or a suitably modified and / or stabilized artificial mRNA.

[0193] According to some implementation methods, mRNA can therefore serve as " Stabilization "Artificial nucleic acids or stabilized coding nucleic acids are provided, namely nucleic acids that exhibit improved in vivo degradation resistance and / or improved in vivo stability, and / or improved in vivo translation efficiency. Specific suitable modifications / adjustments for stabilizing nucleic acids in this context are described below."

[0194] mRNA can be codon-optimized. In some embodiments, the mRNA contains at least one codon-modified coding sequence. In some embodiments, the coding sequence of the mRNA is a codon-modified coding sequence. Suitably, the amino acid sequence encoded by the codon-modified coding sequence is not modified compared to the amino acid sequence encoded by the corresponding wild-type or reference coding sequence. Suitably, the mRNA can be codon-optimized for expression in human cells.

[0195] “ Codon optimization "This refers to the idea that modifications to codon usage can increase the translation efficiency and / or half-life of nucleic acids." (The term "" is used in this context.) Codon-modified coding sequence "This pertains to coding sequences that differ from the corresponding wild-type or reference coding sequence by at least one codon (a nucleotide triplet encoding one amino acid). Suitably, in the context of this invention, codon-modified coding sequences can exhibit improved in vivo degradation resistance and / or improved in vivo stability, and / or improved in vivo translation efficiency. Codon modification in its broadest sense utilizes the degeneracy of the genetic code, where multiple codons can encode the same amino acid and can be used interchangeably (see Table 1 of WO2020002525) to optimize / modify coding sequences for in vivo applications as described herein."

[0196] In some embodiments, the mRNA may be modified such that the C content of at least one coding sequence is increased compared to the C content of the corresponding wild-type or reference coding sequence, suitably maximizing (referred to herein as "C"). C maximizes the encoded sequence Suitablely, the amino acid sequence encoded by the C-maximized coding sequence of the mRNA is not modified compared to the amino acid sequence encoded by the corresponding wild-type or reference coding sequence. The generation of the C-maximized nucleic acid sequence can be suitably performed using the modification method according to WO2015 / 062738. In this context, the disclosure of WO2015 / 062738 is hereby incorporated by reference.

[0197] In some implementations, the mRNA can be modified such that the codons in the at least one coding sequence are adapted for use with human codons (referred to herein as "mRNA"). Human codons use adapted coding sequencesCodons encoding the same amino acid occur at different frequencies in humans. Therefore, the coding sequence of mRNA is suitably modified such that the frequency of codons encoding the same amino acid corresponds to the natural frequency of that codon according to human codon usage. For example, in the case of amino acid Ala, the wild-type or reference coding sequence is suitably adapted such that codon "GCC" is used at a frequency of 0.40, codon "GCT" at a frequency of 0.28, codon "GCA" at a frequency of 0.22, and codon "GCG" at a frequency of 0.10, etc. (see, for example, Table 1 of WO2020002525). Thus, such a procedure (as illustrated for Ala) is applied to each amino acid encoded by the coding sequence of RNA to obtain a sequence adapted to human codon usage.

[0198] In implementation, the mRNA may be modified such that, in the at least one coding sequence, the codon fitness index (CAI) may be increased or suitably maximized (referred to herein as "codon fitness index"). CAI maximizes the encoded sequence In some embodiments, all codons of a wild-type or reference nucleic acid sequence that is relatively rare in humans, for example, are swapped with corresponding codons that are common in humans, where the common codons encode the same amino acid as the relatively rare codons. Suitably, the most common codon is used for each amino acid of the encoded protein (see Table 1 of WO2020002525, where the most common human codons are marked with an asterisk). Suitably, the mRNA contains at least one coding sequence, wherein the codon fitness index (CAI) of the at least one coding sequence is at least 0.5, at least 0.8, at least 0.9, or at least 0.95. In some embodiments, the codon fitness index (CAI) of the at least one coding sequence is 1 (CAI=1). For example, in the case of amino acid Ala, the wild-type or reference coding sequence can be adapted such that the most common human codon "GCC" is always used for that amino acid. Thus, such a procedure (as illustrated for Ala) can be applied to each amino acid encoded by the coding sequence of the mRNA to obtain a coding sequence with maximized CAI.

[0199] In some implementations, the mRNA may be modified, wherein the G / C content of at least one coding sequence may be modified (or optimized) compared to the G / C content of the corresponding wild-type or reference coding sequence (referred to herein as "G / C content"). G / C content modification code sequence In this context, the term " G / C optimization "or" G / C content modificationThis relates to nucleic acids containing a modified number of guanosine and / or cytidine nucleotides, suitably increased compared to the corresponding wild-type or reference coding sequence. Such an increase can be achieved by replacing codons containing adenosine or thymidine nucleotides with codons containing guanosine or cytidine nucleotides. Suitably, nucleic acid sequences with increased G / C content are more stable or exhibit better expression than sequences with increased A / U content. Suitably, the amino acid sequence encoded by the coding sequence modified by the G / C content of the mRNA is not modified compared to the amino acid sequence encoded by the corresponding wild-type or reference sequence. In some embodiments, the G / C content of the coding sequence of the nucleic acid is increased by at least 10%, 20%, 30%, and suitably at least 40% compared to the G / C content of the coding sequence of the corresponding wild-type or reference nucleic acid sequence. The generation of the G / C content-optimized mRNA sequence can be performed using the method according to WO2002 / 098443. In this context, the disclosure of WO2002 / 098443 is included in the present invention in its entirety.

[0200] In some embodiments, the mRNA can be modified by altering the number of A and / or U nucleotides in the nucleic acid sequence relative to the original nucleic acid sequence (e.g., wild-type or reference sequence). In some embodiments, such A / U alterations are made to modify the retention time of individual nucleic acids in an aqueous composition to (i) allow co-purification using HPLC methods and / or allow analysis of the resulting nucleic acid composition. Such methods are described in detail in published PCT application WO2019092153A1. Claims 1 to 70 of WO2019092153A1 are hereby incorporated by reference.

[0201] In some implementations, the modified RNA sequence is selected from C-maximized coding sequences, CAI-maximized coding sequences, human codon usage-adapted coding sequences, G / C content modified (or optimized) sequences, A / U alterations, or any combination thereof.

[0202] In some embodiments, the G / C content of the RNA sequence is at least about 45%, 50%, 55%, or 60%. In specific embodiments, the G / C content of the RNA sequence is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.

[0203] Suitablely, when transfected into mammalian host cells, the mRNA containing the modified sequence is stable for 12-18 hours, or longer than 18 hours, for example, 24, 36, 48, 60, 72 or longer, and is capable of being expressed by mammalian host cells (e.g., muscle cells).

[0204] Suitably, when transfected into mammalian host cells, the mRNA containing the modified RNA sequence is translated into protein, wherein the amount of protein is at least equivalent to, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 100%, or at least 200% or more.

[0205] In some implementations, the mRNA contains at least one poly(N) sequence, such as at least one poly(A) sequence, at least one poly(U) sequence, at least one poly(C) sequence, or a combination thereof.

[0206] In one implementation, the mRNA contains at least one poly(A) sequence. Suitably, a poly(A) tail (e.g., about 30 or more adenosine residues) may be attached to the 3' end of the RNA to increase its half-life.

[0207] As used in this article, the term " poly(A) sequence " poly(A) tail "or" 3'-poly(A) tail "This will be recognized and understood by those skilled in the art, and is intended, for example, to refer to an adenosine nucleotide sequence of up to about 1000 adenosine nucleotides typically located at the 3'-end of a linear RNA (or circular RNA). In some embodiments, the poly(A) sequence is substantially homogeneous, for example, a poly(A) sequence of about 100 adenosine nucleotides is substantially 100 nucleotides in length. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide different from the adenosine nucleotide, for example, a poly(A) sequence of about 100 adenosine nucleotides may be longer than 100 nucleotides (comprising 100 adenosine nucleotides and the at least one nucleotide or nucleotide stretch different from the adenosine nucleotide)."

[0208] The poly(A) sequence may comprise about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. In some embodiments, the length of the poly(A) sequence may be at least about, or even more than about 10, 50, 64, 75, 100, 200, 300, 400, or 500 adenosine nucleotides.

[0209] In some embodiments, the mRNA comprises at least one poly(A) sequence comprising about 30 to about 200 adenosine nucleotides. In some embodiments, the poly(A) sequence comprises about 64 adenosine nucleotides (A64). In other embodiments, the poly(A) sequence comprises about 100 adenosine nucleotides (A100). In other embodiments, the poly(A) sequence comprises about 150 adenosine nucleotides.

[0210] In other embodiments, the mRNA used herein comprises at least one poly(A) sequence comprising about 100 adenosine nucleotides, wherein the poly(A) sequence is interrupted by non-adenosine nucleotides, suitably by 10 non-adenosine nucleotides (A30-N10-A70).

[0211] As defined herein, a poly(A) sequence can be located directly at the 3' end of the mRNA. In some embodiments, the 3'-terminal nucleotide (i.e., the last 3'-terminal nucleotide in the polynucleotide chain) is the 3'-terminal A nucleotide of the at least one poly(A) sequence. The term "..." Located directly at the 3' end "This should be understood as being located exactly at the 3' end; in other words, the 3' end of the nucleic acid consists of a poly(A) sequence terminated by an A nucleotide."

[0212] In one embodiment, the mRNA comprises a poly(A) sequence of at least 70 adenosine nucleotides, preferably at least 70 consecutive adenosine nucleotides, wherein the 3'-terminal nucleotide is an adenosine nucleotide.

[0213] In one embodiment, the poly(A) sequence of the nucleic acid is obtained from a DNA template during in vitro transcription of RNA. In other embodiments, the poly(A) sequence is obtained in vitro by conventional chemical synthesis methods without transcription from a DNA template. In other embodiments, the poly(A) sequence is generated by enzymatic polyadenylation of RNA (after in vitro transcription of RNA) using commercially available polyadenylation kits and appropriate protocols known in the art, or alternatively, by using an immobilized poly(A) polymerase, for example, using the methods and means described in WO2016174271.

[0214] In one implementation, the mRNA contains at least one poly(C) sequence.

[0215] As used in this article, the term " poly(C) sequence"This refers to a cytidine nucleotide sequence of up to about 200 cytidine nucleotides. In embodiments, the poly(C) sequence comprises about 10 to about 200 cytidine nucleotides, about 10 to about 100 cytidine nucleotides, about 20 to about 70 cytidine nucleotides, about 20 to about 60 cytidine nucleotides, or about 10 to about 40 cytidine nucleotides. In one embodiment, the poly(C) sequence comprises about 30 cytidine nucleotides."

[0216] In some embodiments, the mRNA comprises a poly(A) tail sequence, preferably containing 30 to 200 adenosine nucleotides, and / or at least one poly(C) sequence, preferably containing 10 to 40 cytidine nucleotides.

[0217] In some implementations, the mRNA contains at least one histone stem-loop (hSL) or histone stem-loop structure.

[0218] the term" Histone stem-loop "(abbreviated as "hSL" in, for example, sequence listings) refers to nucleic acid sequences that form stem-loop secondary structures that are primarily found in histone mRNAs."

[0219] Histone stem-loop sequences / structures may suitably be selected from histone stem-loop sequences disclosed in WO2012019780, the disclosure relating to which is incorporated herein by reference. Histone stem-loop sequences that may be used may be derived from formula (I) or formula (II) of WO2012019780. According to other embodiments, the mRNA comprises at least one histone stem-loop sequence derived from at least one of formula (Ia) or formula (IIa) of patent application WO2012019780.

[0220] In other implementations, the mRNA does not contain hSL as defined herein.

[0221] mRNA can be modified by adding a 5'-cap structure, which appropriately stabilizes the RNA and / or enhances the expression of the encoded antigen and / or (after administration to the subject) reduces stimulation of the innate immune system.

[0222] As used in this article, the term " 5'-cap structure "will be recognized and understood by those skilled in the art, and is intended, for example, to refer to 5'-modified nucleotides located at the 5'-terminus of RNA (e.g., mRNA), particularly guanine nucleotides."

[0223] For example, the 5' end of mRNA can be capped with a modified ribonucleotide or a derivative thereof having the structure m7G(5')ppp(5')N (cap 0 structure), which can be incorporated during RNA synthesis or enzymatically engineered posttranscriptionally (e.g., by using a vaccinia virus capping enzyme (VCE) composed of mRNA triphosphatase, guanylate transferase, and guanine-7-methyltransferase, which catalyzes the construction of the N7-monomethylated cap 0 structure). The cap 0 structure plays an important role in maintaining the stability of RNA molecules and translation efficiency. The 5' cap of the mRNA molecule can be further modified with a 2'-O-methyltransferase to produce a cap 1 structure (m7Gppp[m2'-O]N), which can further increase translation efficiency.

[0224] In some implementations, the 5'-cap structure is linked to the RNA via a 5'-5'-triphosphate bond.

[0225] Possible suitable 5'-cap structures are cap0 (methylation of the first nucleobase, e.g., m7GpppN), cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), cap3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), cap4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analogs), modified ARCA (e.g., phosphate thioester modified ARCA), inosine, N1-methylguanosine, 2'-fluoroguanosine, 7-deazoguanosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine, and 2-azidoguanosine.

[0226] In some embodiments, the mRNA includes a 5' cap, preferably m7G, cap0, cap1, cap2, a modified cap0 or a modified cap1 structure, suitably a 5'-cap1 structure.

[0227] 5'-cap structures (such as cap0 or cap1) can be formed during chemical RNA synthesis or during in vitro transcription of RNA using cap analogs (co-transcription with capping).

[0228] As used in this article, the term " Hat-like objects"Will be recognized and understood by those skilled in the art, and is intended, for example, to refer to a non-polymerizable dinucleotide or trinucleotide that has a cap function (because when incorporated into the 5'-terminus of a nucleic acid molecule, it facilitates translation or localization, and / or prevents degradation of the nucleic acid molecule, particularly RNA). Non-polymerizable means that the cap analog will only be incorporated into the 5'-terminus because it does not have a 5' triphosphate and therefore cannot be extended in the 3'-direction by template-dependent polymerases, particularly by template-dependent RNA polymerases. Examples of cap analogs include, but are not limited to, chemical structures selected from the group consisting of: m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), and dimethylated symmetrical cap analogs (e.g., m7GppG). pm7G) or anti-reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives). Other cap analogs have been previously described (WO2008016473, WO2008157688, WO2009149253, WO2011015347 and WO2013). 059475). In this context, other suitable cap analogues are described in WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017 / 053297, WO2017066782, WO2018075827 and WO2017066797, the disclosures relating to cap analogues of which are incorporated herein by reference.

[0229] In some embodiments, modified cap1 structures are generated using trinucleotide cap analogs disclosed in WO2017053297, WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017066782, WO2018075827, and WO2017066797. In particular, any cap structure that can be derived from the structure disclosed in claims 1-5 of WO2017053297 can be suitably used for co-transcription to generate modified cap1 structures. Furthermore, any cap structure that can be derived from the structure defined in claim 1 or claim 21 of WO2018075827 can be suitably used for co-transcription to generate modified cap1 structures.

[0230] In some implementations, the mRNA contains a cap1 structure.

[0231] In some implementations, the 5'-cap structure may be co-transcribed into a RNA in vitro transcription reaction as defined herein, using a trinucleotide cap analogue as defined herein.

[0232] In some implementations, the cap1 structure of the mRNA is formed using co-transcriptional capping with the trinucleotide cap analogs m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG. In this context, a suitable cap1 analog is m7G(5')ppp(5')(2'OMeA)pG.

[0233] In other embodiments, the cap1 structure of the mRNA is formed using co-transcriptional capping with the trinucleotide cap analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0234] In other embodiments, the cap0 structure of the mRNA used herein is formed using co-transcriptional capping with the cap analog 3'OMe-m7G(5')ppp(5')G.

[0235] In other embodiments, the 5'-cap structure is formed by enzymatic capping using a capping enzyme (e.g., vaccinia virus capping enzyme and / or cap-dependent 2'-O methyltransferase) to produce cap0, cap1, or cap2 structures. The 5'-cap structure (cap0 or cap1) can be added using the methods and means disclosed in WO2016193226, employing immobilized capping enzymes and / or cap-dependent 2'-O methyltransferases.

[0236] To determine the presence / absence of the cap0 or cap1 structure, a capping assay can be used as described in published PCT application WO2015101416, particularly claims 27 to 46 of published PCT application WO2015101416. Other capping assays that can be used to determine the presence / absence of the cap0 or cap1 structure of RNA are described in PCT / EP2018 / 08667 or published PCT applications WO2014152673 and WO2014152659.

[0237] In some embodiments, the mRNA contains an m7G(5')ppp(5')(2'OMeA) cap structure. In such embodiments, the mRNA contains a 5'-terminal m7G cap and additional methylation of the ribose of the adjacent nucleotide of m7GpppN, in this case, a 2'O-methylated adenosine. In some embodiments, approximately 70%, 75%, 80%, 85%, 90%, or 95% of the RNA (material) contains such a cap1 structure as determined using a capping assay.

[0238] In other embodiments, the mRNA used herein comprises an m7G(5')ppp(5')(2'OMeG) cap structure. In such embodiments, the mRNA comprises a 5'-terminal m7G cap, and additional methylation of the ribose of the adjacent nucleotide, in this case, 2'O-methylated guanosine. In some embodiments, approximately 70%, 75%, 80%, 85%, 90%, or 95% of the coding RNA (material) comprises such a cap1 structure as determined using a capping assay.

[0239] Therefore, the first nucleotide of the mRNA sequence, i.e. the nucleotide downstream of the m7G(5')ppp structure, can be 2'O methylated guanosine or 2'O methylated adenosine.

[0240] Suitablely, the mRNA used herein contains a ribosome binding site, also known as a Kozak sequence. In some embodiments, the A / U (A / T) content in the ribosome binding site environment of the mRNA used herein may be increased compared to the A / U (A / T) content in the ribosome binding site environment of the corresponding wild-type or reference nucleic acid. This modification (increased A / U (A / T) content around the ribosome binding site) increases the efficiency of ribosome binding to mRNA. Efficient binding of ribosomes to the ribosome binding site, in turn, leads to efficient translation of mRNA.

[0241] In some implementations, the mRNA contains at least one heterountranslated region (UTR), such as a 5' UTR and / or a 3' UTR.

[0242] the term" Non-translated area "or" UTR "or" UTR components "UTR will be recognized and understood by those skilled in the art, and is intended, for example, to refer to a portion of a nucleic acid molecule typically located at the 5' or 3' of a coding sequence. A UTR is not translated into a protein. A UTR can be a portion of a nucleic acid (e.g., DNA or RNA). A UTR can contain elements used to control gene expression, also known as regulatory elements. Such regulatory elements can be, for example, ribosome binding sites, miRNA binding sites, promoter elements, etc."

[0243] In some implementations, the mRNA contains a protein-coding region (“mRNA”). Encoded sequence "or" cdsThe 3'-UTR and / or 5'-UTR are included. Notably, the UTR may contain regulatory sequence elements that determine the turnover, stability, and localization of nucleic acids (e.g., RNA). Furthermore, the UTR may contain sequence elements that enhance translation. In medical applications of nucleic acid sequences (including DNA and RNA), the translation of nucleic acids into at least one peptide or protein is crucial for therapeutic efficacy. Certain combinations of 3'-UTR and / or 5'-UTR can enhance the expression of the coding sequence encoding the operably linked peptide or protein of the present invention. Nucleic acid molecules containing UTR combinations are advantageously capable of achieving rapid and transient expression of antigenic peptides or proteins, suitably intramuscularly, after administration to a subject. Therefore, mRNAs containing certain combinations of 3'-UTR and / or 5'-UTR as provided herein are particularly suitable for administration as vaccines or therapeutic agents, especially for administration to the muscle, dermis, or epidermis of a subject.

[0244] In some embodiments, the mRNA comprises at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR. The heterologous 5'-UTR or 3'-UTR may be derived from a naturally occurring gene or may be synthetically engineered. In embodiments, the mRNA comprises at least one coding sequence as defined herein, which is operatively linked to at least one (heterologous) 3'-UTR and / or at least one (heterologous) 5'-UTR.

[0245] In some implementations, the mRNA contains at least one heterologous 3'-UTR.

[0246] the term" 3' - Non-translation area "or" 3'-UTR "or" 3'-UTR element "This will be recognized and understood by those skilled in the art, and is intended, for example, to refer to a portion of a nucleic acid molecule located at the 3' (i.e., downstream) of a coding sequence that is not translated into a protein. The 3'-UTR can be a portion of a nucleic acid (e.g., DNA or RNA) located between the coding sequence and (optionally) the terminal poly(A) sequence. The 3'-UTR may contain elements for controlling gene expression, also known as regulatory elements. Such regulatory elements can be, for example, ribosome binding sites, miRNA binding sites, etc."

[0247] In some implementations, the mRNA contains a 3'-UTR, which may be derived from a gene associated with RNA having an enhanced half-life (i.e., providing stable RNA).

[0248] In some implementations, the 3'-UTR includes one or more of the following: a polyadenylation signal, a protein binding site that affects nucleic acid stability or localization in the cell, or one or more miRNAs or miRNA binding sites.

[0249] In an embodiment, the mRNA comprises at least one heterologous 3'-UTR, wherein the at least one heterologous 3'-UTR comprises a nucleic acid sequence of a 3'-UTR derived from or selected from the following genes: PSMB3, ALB7, α-globin (referred to as "..."). muag (CASP1, COX6B1, GNAS, NDUFA1, and RPS9), or homologs, fragments, or variants of any of these genes.

[0250] In some embodiments, the mRNA comprises or consists of a nucleic acid sequence containing or composed of a 3'-UTR derived from the genes selected from PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or homologs, fragments, or variants of any of these genes.

[0251] The nucleic acid sequence in this context can be derived from the published PCT application WO2019077001A1, particularly claim 9 of WO2019077001A1. The corresponding 3'-UTR sequence of claim 9 of WO2019077001A1 is hereby incorporated by reference.

[0252] In some embodiments, the mRNA comprises a 3'-UTR as described in WO2016107877, the disclosure of the 3'-UTR sequence in WO2016107877 being incorporated herein by reference. Suitable 3'-UTRs are SEQ ID NOs: 1-24 and SEQ ID NOs: 49-318 of WO2016107877, or fragments or variations thereof. In other embodiments, the mRNA comprises a 3'-UTR as described in WO2017036580, the disclosure of the 3'-UTR sequence in WO2017036580 being incorporated herein by reference. Suitable 3'-UTRs are SEQ ID NOs: 152-204 of WO2017036580, or fragments or variations thereof. In other embodiments, the mRNA comprises a 3'-UTR as described in WO2016022914, the disclosure of the 3'-UTR sequence in WO2016022914 of which is incorporated herein by reference. Particularly suitable 3'-UTRs are nucleic acid sequences of SEQ ID NO: 20-36 according to WO2016022914, or fragments or variants of such sequences.

[0253] In some implementations, the mRNA contains at least one heterologous 5'-UTR.

[0254] the term" 5' - Non-translation area "or" 5'-UTR "or" 5'-UTR element"5'-UTR" will be recognized and understood by those skilled in the art, and is intended, for example, to refer to a portion of a nucleic acid molecule located at the 5' (i.e., "upstream") of a coding sequence that is not translated into a protein. A 5'-UTR can be a portion of a nucleic acid located at the 5' of a coding sequence. Typically, a 5'-UTR begins at a transcription start site and ends before the start codon of the coding sequence. A 5'-UTR may contain elements for controlling gene expression, also known as regulatory elements. Such regulatory elements can be, for example, ribosome binding sites, miRNA binding sites, etc. A 5'-UTR can be posttranscribed, for example by enzymatic or posttranscriptional addition of a 5'-cap structure (e.g., for mRNA as defined herein).

[0255] In some implementations, the mRNA contains a 5'-UTR, which may be derived from a gene associated with RNA having an enhanced half-life (i.e., providing stable RNA).

[0256] In some implementations, the 5'-UTR includes one or more of the following: a protein binding site that affects RNA stability or RNA localization in the cell, or a binding site for one or more miRNAs.

[0257] In an embodiment, the mRNA comprises at least one heterologous 5'-UTR, wherein the at least one heterologous 5'-UTR comprises a nucleic acid sequence derived from or selected from the 5'-UTRs of the following genes: HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or homologs, fragments, or variants of any of these genes.

[0258] The nucleic acid sequence in this context may be selected from the published PCT application WO2019077001A1, particularly claim 9 of WO2019077001A1. The corresponding 5'-UTR sequence of claim 9 of WO2019077001A1 is hereby incorporated by reference (e.g., SEQ ID NO: 1-20 of WO2019077001A1, or fragments or variants thereof).

[0259] In some embodiments, the mRNA comprises a 5'-UTR as described in WO2013143700, the disclosure of the 5'-UTR sequence in WO2013143700 being incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences derived from SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421, and SEQ ID NO: 1422 of WO2013143700, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 5'-UTR as described in WO2016107877, the disclosure of the 5'-UTR sequence in WO2016107877 being incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences according to SEQ ID NOs: 25-30 and SEQ ID NOs: 319-382 of WO2016107877, or fragments or variants of these sequences. In other embodiments, the mRNA comprises a 5'-UTR as described in WO2017036580, the disclosure of the 5'-UTR sequence in WO2017036580 being incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences of SEQ ID NOs: 1-151 according to WO2017036580, or fragments or variants of such sequences. In other embodiments, the mRNA comprises a 5'-UTR as described in WO2016022914, the disclosure of the 5'-UTR sequence in WO2016022914 being incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences of SEQ ID NOs: 3-19 according to WO2016022914, or fragments or variants of such sequences.

[0260] In some embodiments, the mRNA comprises a nucleic acid sequence derived from the 5'-UTR of HSD17B4 or a heterologous 5'-UTR thereof, and at least one nucleic acid sequence derived from the 3'-UTR of PSMB3 or a heterologous 3'-UTR thereof.

[0261] In one implementation, the mRNA comprises from 5' to 3': i) 5'-cap1 structure; ii) 5'-UTR derived from the 5'-UTR of the HSD17B4 gene; iii) The encoded sequence; iv) 3'-UTR derived from the 3'-UTR of the PSMB3 gene; v) Optionally, histone stem-loop sequences; and vi) contains a poly(A) sequence of approximately 100 A nucleotides, wherein the 3' terminal nucleotide of the RNA is adenosine.

[0262] Chemical modification In some implementations, the RNA (suitably mRNA) contains a coding sequence consisting only of G, C, A, and U nucleotides, and therefore does not contain modified nucleotides (except for 5' cap structures (cap0, cap1, cap2)).

[0263] In some implementations, the RNA (suitably mRNA) is modified RNA (suitably mRNA), wherein the modification refers to chemical modifications that include main chain modifications as well as sugar or base modifications.

[0264] Modified RNA (suitably mRNA) may contain one or more nucleotide analogs or modified nucleotides (nucleotide analogs / modifications, such as backbone modifications, sugar modifications, or base modifications). As used herein, " Nucleotide analogues "or" Modified nucleotides "" refers to nucleotides containing one or more chemical modifications (e.g., substitutions) in or on a nitrogenous base of a nucleoside (e.g., cytosine (C), thymine (T), or uracil (U), adenine (A), or guanine (G)) and / or one or more chemical modifications in or on a phosphate ester of the backbone. Nucleotide analogs may contain other chemical modifications in or on a sugar moiety of a nucleoside (e.g., ribose, modified ribose, hexaglycose analogs, or open-chain sugar analogs) or phosphate ester. The preparation of nucleotides and modified nucleotides, as well as nucleosides, is well known in the art, see the following references: U.S. Patent Nos. 4373071, 4458066, 4500707, 4668777, 4973679, 5047524, 5132418, 5153319, 5262530, 5700642. Many modified nucleosides and modified nucleotides are commercially available.

[0265] As described herein, backbone modification is a modification in which the phosphate esters of the nucleotide backbone of RNA (suitably mRNA) are chemically modified. As described herein, sugar modification is a chemical modification of the sugars in the nucleotides of RNA (suitably mRNA). Furthermore, as described herein, base modification is a chemical modification of the base portions of the nucleotides of RNA (suitably mRNA). In this context, nucleotide analogs or modifications are suitably selected from nucleotide analogs applicable to transcription and / or translation.

[0266] In some implementations, the RNA (suitably mRNA) contains at least one chemical modification.

[0267] In some embodiments, the chemical modification is selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine and 5-methoxyuridine, more preferably pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), and even more preferably N1-methylpseudouridine (m1ψ).

[0268] In some implementations, substantially all (e.g., substantially 100%) of the uracil in the coding sequence of the RNA (suitably mRNA) is chemically modified, preferably at position 5 of the uracil.

[0269] In some embodiments, the RNA (suitably mRNA) contains chemical modifications as uridine modifications, preferably wherein 100% of the uridine sites in the mRNA are modified.

[0270] Incorporating modified nucleotides (such as pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine and / or 5-methoxyuridine) into the coding sequence of the RNA (suitably mRNA) used herein may be advantageous because unwanted innate immune responses (after administration of the coding mRNA or vaccine) can be modulated or reduced (if necessary).

[0271] In some embodiments, the coding sequence of the RNA (suitably mRNA) comprises at least one modified nucleotide selected from pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), suitably wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides, optionally wherein all uracil nucleotides are replaced by pseudouridine (Ψ) nucleotides and / or N1-methylpseudouridine (m1Ψ) nucleotides.

[0272] In some embodiments, the RNA (suitably mRNA) does not contain the site of the N1-methylpseudouridine (m1Ψ) substitution. In other embodiments, the RNA (suitably mRNA) used herein does not contain the sites of pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine substitutions.

[0273] In some embodiments, the chemical modification is N1-methylpseuuridine and / or pseudouridine.

[0274] In the context of nucleic acid-based vaccine or therapeutic agent production, it may be necessary to provide GMP-grade nucleic acids, such as GMP-grade RNA or DNA. GMP-grade RNA or DNA can be produced using manufacturing processes approved by regulatory authorities. Therefore, in some embodiments, RNA production is carried out under current Good Manufacturing Practices (GMP), with various quality control steps implemented at the DNA and RNA levels, suitably in accordance with WO2016180430. In embodiments, the RNA (suitably mRNA) of the present invention is GMP-grade RNA.

[0275] RNA synthesis In some embodiments, RNA (suitably mRNA) can be prepared using any method known in the art, including chemical synthesis (e.g., solid-phase RNA synthesis) and in vitro methods (e.g., in vitro RNA transcription reaction).

[0276] Appropriately, the RNA (appropriately mRNA) used in this article is RNA transcribed in vitro.

[0277] the term" RNA in vitro transcription "or" In vitro transcriptionThis involves the process of RNA synthesis in a cell-free system (in vitro). RNA can be obtained through DNA-dependent in vitro transcription of a suitable DNA template, which can be a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter used to control the in vitro transcription of RNA can be any promoter of any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, SP6, or Syn5. RNA polymerase. In one embodiment of the invention, the DNA template is linearized with a suitable restriction endonuclease prior to in vitro RNA transcription. The reagents used in in vitro RNA transcription typically include: a DNA template (linearized plasmid DNA or PCR product) having a promoter sequence having a high binding affinity for its corresponding RNA polymerase (e.g., a phage-encoded RNA polymerase (T7, T3, SP6, or Syn5)); ribonucleoside triphosphates (NTPs) of four bases (adenine, cytosine, guanine, and uracil); optionally, cap analogs as defined herein; optionally, other modified nucleotides as defined herein; and a DNA-dependent RNA polymerase (e.g., T7, T3, SP6, or Syn5) capable of binding to the promoter sequence within the DNA template. RNA polymerase); optionally, an RNase inhibitor for inactivating any potentially contaminating ribonuclease (RNase); optionally, a pyrophosphatase for degrading pyrophosphate (which may inhibit in vitro RNA transcription); MgCl2 for supplying Mg2+ ions as a polymerase cofactor; a buffer (TRIS or HEPES) for maintaining a suitable pH, which may also contain an antioxidant (e.g., DTT), and / or an optimal concentration of a polyamine (e.g., spermidine), such as a buffer system containing TRIS-citrate as disclosed in WO2017109161.

[0278] In embodiments, the nucleotide mixture for in vitro transcription of RNA may additionally contain modified nucleotides as defined herein. In this context, suitable modified nucleotides may be particularly selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine. In embodiments, uracil nucleotides in the nucleotide mixture are (partially or completely) replaced with pseudouridine (ψ) and / or N1-methylpseudouridine (m1ψ) to obtain modified RNA.

[0279] In other embodiments, the nucleotide mixture for in vitro transcription of RNA does not contain modified nucleotides as defined herein. In some embodiments, the nucleotide mixture for in vitro transcription of RNA contains only G, C, A, and U nucleotides, and optionally cap analogs as defined herein.

[0280] In some implementations, the nucleotide mixture used for in vitro transcription of RNA (i.e., the proportion of each nucleotide in the mixture) can be optimized for a given RNA sequence, suitably as described in WO2015188933.

[0281] In this context, in vitro transcription has been performed in the presence of sequence-optimized nucleotide mixtures and optional cap analogs.

[0282] RNA purification Suitable RNA (or mRNA) is purified RNA (or mRNA).

[0283] As used in this article, the term " Purified RNA (or mRNA) "This should be understood as RNA with a higher purity than the starting material (e.g., RNA transcribed in vitro) after certain purification steps (e.g., HPLC, TFF, Oligo d(T) purification, precipitation steps). Typical impurities that are essentially not present in purified RNA include peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription, such as RNA polymerase, RNase, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, aborted RNA sequences, RNA fragments (short double-stranded RNA fragments, aborted sequences, etc.), free nucleotides (modified nucleotides, conventional NTPs, cap analogs), template DNA fragments, buffer components (HEPES, TRIS, MgCl2), etc. Other potential impurities that can be derived, for example, from fermentation procedures include bacterial impurities (bioload, bacterial DNA) or impurities derived from purification procedures (organic solvents, etc.). Therefore, in this respect, " RNA purity "Ideally, it should be as close to 100% as possible. For RNA purity, it is also ideal for the amount of full-length RNA transcript to be as close to 100% as possible. Therefore, as used in this article," Purified RNA (or mRNA) The purity exceeds 75%, 80%, 85%, very specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and most advantageously 99% or higher. Purity can be determined, for example, by analytical HPLC, where the percentages provided above correspond to the ratio between the peak area of ​​the target RNA and the total area of ​​all peaks representing byproducts. Alternatively, purity can be determined, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0284] In some embodiments, RNA is purified using RP-HPLC, suitably using reversed-phase high-performance liquid chromatography (RP-HPLC) with a macroporous styrene / divinylbenzene column (e.g., 30 µm particle size, 4000 Å pore size), and additionally using a filter cartridge with a cellulose-based membrane having a molecular weight cutoff of approximately 100 kDa. RNA can be purified, particularly using PUREMESSENGER (CureVac, Germany). Purification was performed according to RP-HPLC (as described in WO2008077592) and / or tangential flow filtration (as described in WO2016193206) and / or oligo d(T) purification (see WO2016180430).

[0285] In some implementations, RNA (suitably mRNA) is purified by RP-HPLC and / or TFF to remove double-stranded RNA, uncapped RNA, and / or RNA fragments.

[0286] Double-stranded RNA, formed as a byproduct during, for example, RNA in vitro transcription, can lead to the induction of innate immune responses, particularly IFNα, which is a major factor in inducing fever in vaccinated subjects—an undesirable side effect. Current techniques for dsRNA immunoblotting (e.g., by dot blot, serological-specific electron microscopy (SSEM), or ELISA) are used to detect dsRNA material from nucleic acid mixtures and determine its size.

[0287] In this embodiment, the RNA (suitably mRNA) contains approximately 5%, 10%, or 20% less double-stranded RNA byproducts than RNA (suitably mRNA) that has not been purified by RP-HPLC and / or TFF.

[0288] In some embodiments, RNA purified by RP-HPLC and / or TFF (suitably mRNA) contains approximately 5%, 10%, or 20% less double-stranded RNA byproducts than RNA purified by OligodT, precipitated, filtered, and / or AEX (suitably mRNA).

[0289] Suitably, the aqueous composition is a therapeutic composition. In one embodiment, the therapeutic composition is an immunogenic composition, suitably a vaccine composition.

[0290] the term" Immunogen "or" Immunogenicity "will be recognized and understood by those skilled in the art, and is intended, for example, to refer to compounds capable of stimulating / inducing (adaptive) immune responses. Immunogens may be peptides, polypeptides, or proteins."

[0291] the term" Immune response"will be recognized and understood by those skilled in the art, and is intended, for example, to refer to a specific response of the adaptive immune system to a particular antigen (so-called specific or adaptive immune response) or a non-specific response of the innate immune system (so-called non-specific or innate immune response) or a combination thereof."

[0292] The therapeutic composition according to the invention can be administered via various suitable routes, including parenteral, intramuscular, intradermal, intranasal, or subcutaneous administration. Suitably, the therapeutic composition is suitable for intramuscular administration to a subject.

[0293] Subjects intended to administer the therapeutic composition include, but are not limited to, mammals, including humans and / or other primates, as well as commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys. Suitably, the therapeutic composition is intended for administration to human subjects.

[0294] Suitably, the RNA molecule is mRNA encoding a protein of interest. In one embodiment, the mRNA encoding the protein of interest is a non-replicating mRNA. In another embodiment, the mRNA encoding the protein of interest is either a non-replicating mRNA or a self-replicating mRNA. Suitably, the protein of interest is an antigen.

[0295] As used in this article, the term " antigen "As will be recognized and understood by those skilled in the art, and is intended, for example, to refer to substances that can be recognized by the immune system (e.g., by the adaptive immune system) and are capable of triggering an antigen-specific immune response (e.g., through the formation of antibodies and / or antigen-specific T cells as part of an adaptive immune response). Typically, an antigen can be or may contain a peptide or protein that can be presented to T cells by the MHC. Fragments, variants, and derivatives of peptides or proteins containing at least one epitope are also understood to be antigens."

[0296] Suitablely, the protein of interest is an antigen derived from a respiratory virus, such as influenza virus, SARS-CoV-2 virus, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), or human parainfluenza virus (PIV).

[0297] In one embodiment, the protein of interest is an influenza virus antigen selected from hemagglutinin (HA) antigen and neuraminidase (NA) antigen.

[0298] In one implementation, the protein of interest is the SARS-CoV-2 spike protein (S) antigen.

[0299] In one implementation, the protein of interest is the RSV fusion protein (F) antigen.

[0300] In one implementation, the protein of interest is the HMPV fusion protein (F) antigen.

[0301] In one implementation, the protein of interest is the PIV fusion protein (F) antigen.

[0302] In some embodiments, the aqueous composition of the present invention is obtained by first preparing RNA (suitably mRNA) and a delivery system (suitably LNP) in a first buffer system, followed by buffer exchange to obtain an aqueous composition as defined herein (e.g., containing Tris, sugar, phosphate anion, sodium chloride and optional glycerol).

[0303] The intermediate buffer is preferably a phosphate buffer. Preferably, the intermediate buffer is a PBS-sucrose buffer, which preferably contains about 150 mM sucrose, about 75 mM NaCl and about 10 mM Na3PO4.

[0304] Buffer exchange from intermediate buffer to aqueous composition can be performed by dilution. Dilution factors can be in the range of 1:1 to 1:100, suitably 1:1 to 1:10, more preferably 1:2 to 1:5, for example 1:3.

[0305] In another aspect, the present invention provides an aqueous composition for therapeutic purposes, suitably used as a vaccine.

[0306] In another aspect, the present invention provides a method for treating or preventing disease, comprising applying the aqueous composition of the present invention to a patient in need of such treatment.

[0307] In another aspect, the present invention provides a method for treating or preventing infectious diseases, comprising applying the aqueous composition of the present invention to a patient in need of such treatment.

[0308] In another aspect, the present invention provides the use of the aqueous compositions of the present invention in the preparation of pharmaceuticals or vaccines.

[0309] In another aspect, the present invention provides a method for preparing the aqueous composition of the present invention, comprising: a) Provide an initial composition comprising an RNA molecule, preferably mRNA, and a delivery system, preferably LNP. b) Transfer the initial composition to the first buffer system to obtain the intermediate composition. c) Dilute the intermediate composition with a dilution buffer to obtain the aqueous composition of the present invention.

[0310] The first buffer system may be a phosphate buffer, such as PBS buffer. Suitably, the PBS buffer contains sucrose. Suitably, the PBS sucrose buffer contains, preferably, about 150 mM sucrose; preferably, about 75 mM NaCl; and preferably, about 10 mM NaPO4; and has a pH preferably about 7.4.

[0311] The transfer step b) of this method can be performed using filtration, such as ultrafiltration or tangential flow filtration (TFF).

[0312] The dilution in step c) of the method can be performed with a dilution factor in the range of 1:1 to 1:100, suitably 1:1 to 1:10, more suitably 1:2 to 1:5, for example 1:3.

[0313] The dilution buffer contains sugars and Tris as defined herein. In some embodiments, the dilution buffer contains glycerol. In some embodiments, the dilution buffer may contain phosphate anions and / or sodium chloride.

[0314] Further definition For clarity and readability, the following definitions are provided. Any technical features mentioned in connection with these definitions can be interpreted in relation to various and every embodiment of the invention. Further definitions and explanations may be provided in the context of these embodiments.

[0315] Throughout the specification (including the claims), where the context permits, the term " Include (comprising) "and its variants such as " Includes "Should be interpreted as including the stated element (e.g., an integer) or multiple elements (e.g., multiple integers), without necessarily excluding any other element (e.g., multiple integers). Therefore, an aqueous composition "containing" X may consist of only X, or may contain additional substances, such as X+Y."

[0316] Words " basically The use of the word "completely" is not excluded; for example, an aqueous composition that is "substantially free" of Y can be completely free of Y. The word "substantially" may be omitted from the definition of this invention where necessary.

[0317] As used herein, unless the context explicitly indicates otherwise, the singular forms “a,” “an,” and “the” include plural references.

[0318] Unless otherwise specified, methods involving the mixing of two or more components do not require any particular mixing order. Therefore, the components can be mixed in any order. In the presence of three components, two components can be combined with each other, and that combination can then be combined with a third component, and so on.

[0319] In the context of numbers, percentage It should be understood as relative to the total number of the corresponding items. In other cases, and unless the context otherwise indicates, percentages should be understood as weight percentages (wt.-%).

[0320] about The term "about" is used when determinants or values ​​do not need to be identical (i.e., 100% identical). Therefore, "about" means that a determinant or value can deviate from 1% to 20%, for example, 1% to 10%; specifically, deviations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20%. Those skilled in the art will understand that, for example, certain parameters or determinants can vary slightly based on the way the parameters are determined. For example, if a determinant or value is defined herein as having a length of, for example, "about 100 nucleotides," then that length can deviate from 1% to 20%. Therefore, those skilled in the art will understand that in this specific example, the length can deviate from 1 to 20 nucleotides. Thus, a length of "about 100 nucleotides" can encompass sequences ranging from 80 to 120 nucleotides.

[0321] Encoded sequence / encoded region As used herein, the terms "coding sequence" or "coding region" and the corresponding abbreviation "cds" will be recognized and understood by those skilled in the art, and are intended, for example, to refer to a sequence of several nucleotide triplets that can be translated into a peptide or protein. In the context of this invention, a coding sequence can be an RNA sequence consisting of a number of nucleotides divisible by three, beginning with a start codon and, for example, ending with a stop codon.

[0322] Derived from...The term "derived from" as used throughout this specification in the context of nucleic acids means, for a nucleic acid "derived" from (another) nucleic acid, that the nucleic acid derived from (another) nucleic acid shares, for example, at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid from which it was derived. Those skilled in the art will understand that sequence identity is generally calculated for the same type of nucleic acid, i.e., for DNA sequences or for RNA sequences. Therefore, it should be understood that if DNA is "derived" from RNA, or if RNA is "derived" from DNA, then in the first step, the RNA sequence is converted to the corresponding DNA sequence (particularly by replacing uracil (U) with thymine (T) throughout the sequence), or conversely, the DNA sequence is converted to the corresponding RNA sequence (particularly by replacing T with U throughout the sequence). Thereafter, the sequence identity of the DNA sequence or the sequence identity of the RNA sequence is determined. For example, nucleic acids “derived from” nucleic acids also refer to nucleic acids that have been modified compared to the nucleic acids from which they are derived, for example, to facilitate or even further increase RNA stability and / or elongate and / or increase protein production. In the context of amino acid sequences (e.g., antigenic peptides or proteins), the term “derived from” indicates that the amino acid sequence derived from (another) amino acid sequence shares, for example, at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence from which it is derived.

[0323] ExcerptThe term "fragment" as used throughout this specification in the context of nucleic acid sequences (e.g., RNA or DNA) or amino acid sequences can generally refer to a shorter portion of, for example, a full-length nucleic acid or amino acid sequence. Thus, a fragment typically consists of the same sequence as the corresponding segment within the full-length sequence. In the context of this invention, a specific fragment of a sequence consists of consecutive segments of an entity, such as nucleotides or amino acids corresponding to consecutive segments of an entity in a molecule (e.g., a viral protein) from which the fragment is derived, representing at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the total (i.e., full-length) molecule from which the fragment is derived. The term "fragment" as used throughout this specification in the context of proteins or peptides can generally comprise a sequence of a protein or peptide as defined herein, whose N-terminus and / or C-terminus are truncated compared to the amino acid sequence of the original protein. The term "fragment" as used throughout this specification in the context of RNA sequences can generally comprise an RNA sequence whose 5'-terminus and / or 3'-terminus are truncated compared to a reference RNA sequence. Therefore, such truncation can occur at the amino acid level or, correspondingly, at the nucleic acid level. Thus, sequence identity with respect to such fragments as defined herein can refer, for example, to the entire protein or peptide as defined herein, or the entire (encoding) nucleic acid molecule of such protein or peptide. A fragment of a protein or peptide may contain at least one epitope of those proteins or peptides.

[0324] Heterogeneous As used throughout this specification in the context of nucleic acid or amino acid sequences, the term "heterologous" or "heterologous sequence" means a sequence (e.g., RNA, DNA, amino acid) derived from another gene, another allele, or, for example, another species or virus. Two sequences are generally understood to be "heterologous" if they cannot be derived from the same gene or the same allele. That is, although heterologous sequences may be derived from the same organism or virus, they do not appear in the same nucleic acid or protein in nature.

[0325] (Sequence) IdentityThe term "identity," as used throughout this specification in the context of nucleic acid or amino acid sequences, will be recognized and understood by those skilled in the art, and is intended, for example, to refer to the percentage of two sequences that are identical. To determine the percentage of two sequences that are identical, such as nucleic acid sequences or amino acid (aa) sequences as defined herein, such as an aa sequence encoded by a nucleic acid sequence as defined herein, or the aa sequence itself, sequences can be aligned for subsequent comparison with each other. Thus, for example, a position in a first sequence can be compared with a corresponding position in a second sequence. If a position in the first sequence is occupied by the same residue at the same position in the second sequence, the two sequences are identical at that position. If this is not the case, the sequences are different at that position. If an insertion occurs in the second sequence compared to the first sequence, a gap can be inserted in the first sequence to allow for further alignment. If a deletion occurs in the second sequence compared to the first sequence, a gap can be inserted in the second sequence to allow for further alignment. The percentage of two sequences that are identical is a function of the number of identical positions divided by the total number of positions (including those occupied only in one sequence). The percentage of two sequences that are identical can be determined using algorithms, such as those integrated into BLAST programs. Sequence identity can be determined using the EMBOSS Water sequence alignment tool at the EMBL-EBI website (https: / / www.ebi.ac.uk / Tools / psa / emboss_water / ) with parameters of gap open = 12, gap extend = 1, and matrix = BLOSUM62 (for protein sequences) or matrix = full DNA (for DNA / RNA sequences). Alternatively, it can be determined using the EMBOSS Needle sequence alignment tool at the EMBL-EBI website (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ) with default parameters (e.g., gap open = 10, gap extend = 0.5, end gap penalty = no, end gap open = 10, end gap extend = 0.5, and matrix = BLOSUM62 (for protein sequences) or matrix = full DNA (for DNA / RNA sequences)). Unless otherwise stated, when referring to sequence identity with a particular reference sequence in this application, identity is intended to be calculated over the entire length of the reference sequence.

[0326] Nucleic acid, nucleic acid moleculesAs used herein, the terms "nucleic acid" or "nucleic acid molecule" will be recognized and understood by those skilled in the art. The term "nucleic acid" or "nucleic acid molecule" specifically refers to DNA (molecules) or RNA (molecules). This term is used synonymously with the term polynucleotide. For example, a nucleic acid or nucleic acid molecule is a monomer or polymer comprising nucleotides covalently linked to each other by phosphodiester bonds of a sugar / phosphate backbone. The term "nucleic acid" or "nucleic acid molecule" also encompasses modified nucleic acids (molecules), such as DNA or RNA (molecules) that have undergone base modification, sugar modification, or backbone modification as defined herein.

[0327] Nucleic acid sequence, DNA sequence, RNA sequence The terms “nucleic acid sequence,” “DNA sequence,” and “RNA sequence” will be recognized and understood by those skilled in the art, and, for example, refer to a specific and individual sequence of its nucleotide continuity.

[0328] Stabilized RNA The term "stabilized RNA" refers to RNA modified in such a way that it is more stable against disintegration or degradation (e.g., by environmental factors or enzymatic digestion, such as degradation by exonucleases or endonucleases) compared to RNA without such modification. Preferably, in the context of this invention, the stabilized RNA is stabilized in cells (such as prokaryotic or eukaryotic cells, preferably mammalian cells, such as human cells). The stabilization effect can also be exerted extracellularly, for example in buffer solutions, such as aqueous compositions used for storing stabilized RNA.

[0329] (Sequence) variants The term "variant," as used throughout this specification in the context of nucleic acid sequences, will be recognized and understood by one of ordinary skill in the art, and is intended, for example, to refer to a variant of a nucleic acid sequence derived from another nucleic acid sequence. For example, a variant of a nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions, and / or substitutions compared to the nucleic acid sequence of the derived variant. A variant of a nucleic acid sequence may be at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence of the derived variant. A variant is a functional variant, meaning that the variant retains at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence from which it is derived. A "variant" of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% nucleotide identity over a segment of at least 10, 20, 30, 50, 75, or 100 nucleotides of such a nucleic acid sequence.

[0330] As used throughout this specification in the context of proteins or peptides, the term "..." variants"For example, it refers to protein or peptide variants that differ from the original sequence by one or more mutations / substitutions (such as one or more substitutions, insertions, and / or deletions of amino acids). Suitably, these fragments and / or variants have the same or equivalent specific antigenic properties (immunogenic variants, antigenic variants). Insertions and substitutions are particularly possible at sequence positions that do not cause modification of the three-dimensional structure or affect the binding region. Modifications to the three-dimensional structure by insertions or deletions can be readily identified, for example, using CD spectroscopy (circular dichroism spectroscopy). A 'variant' of a protein or peptide may have at least 70%, 75%, or 75% of the amino acid sequence differing from the original sequence by at least 10, 20, 30, 50, 75, or 100 amino acids." 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity. Alternatively, a “variant” of a protein or peptide may have 1 to 20, for example 1 to 10, single-amino acid mutations compared to such a protein or peptide, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, or 20 single-amino acid mutations. For mutations, we indicate or include substitutions, insertions, or deletions. In one embodiment, a variant of a protein comprises a functional variant of the protein, which, in the context of this invention, means that the variant exhibits substantially the same, or at least 40%, 50%, 60%, 70%, 80%, or 90% immunogenicity as the protein from which it is derived.

[0331] Example The following presents specific embodiments illustrating various methods and aspects of the invention. However, the invention should not be limited in scope to the specific embodiments described herein. The following preparations and examples are provided to enable those skilled in the art to more clearly understand and practice the invention. However, the invention is not limited in scope to the exemplary embodiments, which are intended only to illustrate a single aspect of the invention, and functionally equivalent methods are within the scope of the invention. In fact, various modifications to the invention, other than those described herein, will become apparent to those skilled in the art from the foregoing description, drawings, and the following examples. All such modifications fall within the scope of the appended claims.

[0332] Example 1 - Screening of aqueous mRNA-LNP compositions Liquid formulations were prepared by diluting concentrated mRNA-LNP stock solution (originally in PBS sucrose buffer) in formulations containing different excipient contents.

[0333] The concentrated mRNA / LNP stock solution contains mRNAs expressing four influenza virus antigens (HA and NA from Wisconsin H1N1, and HA and NA from Darwin H3N2), encapsulated in lipid nanoparticles (LNPs) containing DSPC, ALC-0315, cholesterol, and ALC-0159 in a molar ratio of approximately 47.4:10.0:40.9:1.7 (DSPC:ALC-0315:cholesterol:ALC-0159). In addition to the antigen-coding sequences, each mRNA construct contains an mRNA cap (Cap1), an HSD17B4 5' UTR, a PSMB3 3' UTR, a histone stem-loop (HSL), and a polyA tail (A100). The antigen-coding sequences were GC-enriched, and all uridine residues were replaced with N1-methylpseudouridine (m1ψ). The mRNA constructs are approximately 1700 nucleotides (NA) and 1900 nucleotides (HA) in length.

[0334] The excipient content of each aqueous composition tested is shown in Table 1.

[0335] Table 1. Composition of the aqueous composition for each test

[0336] The following analytical methods are briefly described for monitoring the quality properties of the tested formulation: mRNA-LNP size (Z-mean) and polydispersity index (PdI) Dynamic light scattering (DLS) is the chosen technique for characterizing and monitoring the size and polydispersity (size distribution) of LNPs in stability by measuring the diffusion of particles moving under Brownian motion and converting it into size and size distribution using the Stokes-Einstein relation. Analysis was performed using the software ZS EXPLORER via Zetasizer Ultra Pro. Data are reported for the diameter (in nm) and polydispersity index for each sample.

[0337] Encapsulation efficiency (EE) mRNA encapsulation efficiency was measured using the RiboGreen assay, which relies on a dye that fluoresces upon binding to single-stranded mRNA. For intact LNPs, dye accessibility is low, therefore only unencapsulated mRNA was detected. To determine the total mRNA concentration, the encapsulated mRNA was released by cleaving the LNPs with detergent (Triton X-100). The ratio of fluorescence intensity before and after Triton addition allowed for calculation of the proportion of encapsulated mRNA payload.

[0338] mRNA integrity and mRNA LEP (late elution peak) ) After extracting mRNA from LNP, reversed-phase ion-pair high-performance liquid chromatography (RP-IP HPLC) was used to assess mRNA integrity and detect potential adduct events (mRNA LEPs) on intact mRNA. The RP-IP HPLC method for mRNA-lipid adducts has been adapted from the paper by Packer et al. (2021, Nature Communications).

[0339] In vitro relative expression assay HEK293T cells were incubated with varying amounts of mRNA-LNP. Cells were harvested 24 h after incubation, and the surface expression of HA and NA proteins was analyzed by flow cytometry. For this purpose, cells were stained with sheep anti-HA and anti-NA influenza sera, and then collected using a secondary anti-sheep FITC conjugated antibody on a MACSQuant® VYB flow cytometer and analyzed using FlowJo software version 10.9.0.

[0340] result 1. Aqueous compositions numbered 1 to 6 Aqueous compositions numbered 1 to 6 contain mRNA encoding four influenza antigens formulated in LNP as described above, and excipients as shown in Table 1. The total mRNA concentration is 0.3 g / L.

[0341] LNP size (z-mean (nm)) and polydispersity index (PdI) were measured at T0, after 8 days at -20°C, and after 14 days at +5°C. The results are shown in Figure 1. The Tris-based matrix performed better than the phosphate- and histidine-based compositions.

[0342] The proportion of LEP was measured by HPLC at T0, after 8 days at -20℃, after 14 days at +5℃, and after 14 days at +25℃. The results are shown in... Figure 2 In the study, Tris-based matrices outperformed phosphate- and histidine-based compositions.

[0343] 2. Aqueous compositions numbered 5 and 12 Aqueous compositions numbered 5 and 12 contain mRNA encoding four influenza antigens formulated in LNP as described above, and the excipients shown in Table 1. The total mRNA concentration is 0.3 g / L.

[0344] mRNA integrity and LEP ratio were monitored over 21 days at +25°C. Results were shown in Figure 3 (mRNA integrity %) and Figure 4 (LEP%)

[0345] 3. Aqueous compositions numbered 1 and 7-10 Aqueous compositions numbered 1 and 7-10 contain mRNA encoding four influenza antigens formulated in LNP as described above, and the excipients shown in Table 1. The total mRNA concentration is 0.032 g / L.

[0346] Encapsulation efficiency (%) was monitored over 90 days at -20°C, +5°C, and -70°C; over 30 days at +25°C; over 1 month at -20°C followed by 2 months at +5°C; and over 14 days at -70°C followed by 2.5 months at -20°C. The results are shown in Figure 5.

[0347] The LNP size (z-mean (nm)) was monitored over 90 days at -20°C, +5°C, and -70°C; over 30 days at +25°C; over 1 month at -20°C followed by 2 months at +5°C; and over 14 days at -70°C followed by 2.5 months at -20°C. The results are shown in Figure 6.

[0348] LNP PdI was monitored over 90 days at -20°C, +5°C, and -70°C; over 30 days at +25°C; for 1 month at -20°C followed by 2 months at +5°C; and for 14 days at -70°C followed by 2.5 months at -20°C. The results are shown in Figure 7.

[0349] The relative expression (%) in vitro was monitored over 30 days at -20℃, +5℃, and +25℃. The results are shown in Figure 8.

[0350] 4. Aqueous compositions numbered 1, 9 and 11 Aqueous compositions numbered 1, 9, and 11 contain mRNA encoding four influenza antigens formulated in LNP as described above, and excipients as shown in Table 1. The total mRNA concentration is 0.032 g / L.

[0351] The LNP size (z-mean (nm) and PdI) were monitored over 90 days at -20℃, +5℃, and +25℃. The results are shown in Figure 9.

[0352] Encapsulation efficiency (%) was monitored over 90 days at -20℃, 50 days at +5℃, and 50 days at +25℃. The results are shown in Figure 10.

[0353] mRNA integrity (%) and LEP (%) were monitored over 50 days at -20℃, +5℃, and +25℃. The results are shown in Figure 11.

[0354] The relative expression (%) in vitro was monitored over 30 days at -20℃, +5℃, and +25℃. The results are shown in Figure 12.

[0355] 5. Aqueous compositions numbered 6, 9 and 12 Aqueous compositions numbered 6, 9, and 12 contain 0.1 g / L mRNA encoding four influenza antigens formulated in LNP as described above, and the excipients shown in Table 1. The total mRNA concentration is 0.1 g / L.

[0356] The LNP size (z-mean (nm) and PdI) were monitored over time at -20℃ (120 days), +5℃ (90 days), and +25℃ (90 days). The results are shown in Figure 13.

[0357] Encapsulation efficiency (%) was monitored over time at -20℃ (120 days), +5℃ (90 days), and +25℃ (90 days). The results are shown in Figure 14.

[0358] mRNA integrity (%) and LEP (%) were monitored over time at -20℃ (120 days), +5℃ (90 days), and +25℃ (90 days). The results are shown in Figure 15.

[0359] Example 2 - In vivo analysis of the formulation stored at 25°C An mRNA-LNP formulation was prepared comprising mRNA constructs encoding four different influenza antigens (HA and NA from Wisconsin H1N1, and HA and NA from Darwin H3N2), encapsulated in lipid nanoparticles (LNPs) containing DSPC, ALC-0315, cholesterol, and ALC-0159 in a molar ratio of approximately 47.4:10.0:40.9:1.7 (DSPC:ALC-0315:cholesterol:ALC-0159). In addition to the antigen-coding sequences, each mRNA construct contained an mRNA cap (Cap1), an HSD17B4 5'UTR, a PSMB3 3'UTR, a histone stem-loop (HSL), and a polyA tail (A100). The antigen-coding sequences were GC-enriched, and all uridine residues were replaced with N1-methylpseudouridine (m1ψ). The mRNA constructs were approximately 1700 nucleotides (NA) and 1900 nucleotides (HA) in length.

[0360] The excipient content of each aqueous composition tested is provided in Table 2.

[0361] Table 2. Composition of the aqueous composition for each test

[0362] Mice were immunized intramuscularly twice on days 0 and 21 with the corresponding mRNA-LNP formulation at a total mRNA dose of 0.5 µg. Animals in the control group were injected with PSN only (lacking mRNA-LNP).

[0363] Use the following analysis methods: Muscle swelling score Muscle swelling was used as an indicator of local tolerance. Swelling and redness of the injected muscle were monitored by visual inspection after each injection. Swelling was scored as follows compared to uninjected muscle: none (score=0), mild (score=1), moderate (score=2), and significant (score=3).

[0364] Mice were intramuscularly immunized on days 0 and 21 with 0.5 µg of the indicated formulation (A (PSN), B (PSN / TSN), and C (PSN / TSG)) stored at -70°C (T0) or at 25°C for 21 days (21d25°C) (n=14). Control animals (n=5) received the PSN matrix (lacking LNP mRNA) intramuscularly on days 0 and 21. Muscle swelling was scored after each immunization and compared to uninjected muscle: none (score=0), mild (score=1), moderate (score=2), and significant (score=3).

[0365] Quantitative analysis of pro-inflammatory cytokines and chemokines in mouse serum According to the manufacturer's instructions, the LEGENDplex cell counting bead assay was used. TM A mouse antiviral response assay panel (13-plex, including CCL2, CCL5, CXCL1, CXCL10, GM-CSF, IFNα, IFNβ, IFNγ, IL-1β, IL-6, IL-10, IL-12p70, and TNF) was used to quantify cytokines and chemokines in serum samples. Microbeads were measured using flow cytometry (Biorad) and analyzed using software (BioLegend).

[0366] Hemagglutinin inhibition (HI) assay The HI assay was used to assess the functional anti-HA antibody response induced by the administered formulation. The HI assay is based on the prevention of viral attachment to erythrocytes in the presence of influenza virus antibodies. Therefore, the presence of antibodies in the serum of vaccinated mice inhibits hemagglutination. The HI assay was performed on individual serum samples obtained three weeks after the first vaccination and two weeks after the second vaccination. Prior to the assay, serum samples were treated with receptor-degrading enzymes (RDEs) to eliminate non-specific inhibitors of hemagglutination, followed by heat inactivation for 30 minutes and pre-adsorption to erythrocytes (RBCs). Mouse serum samples treated with NaCl solution served as negative controls. Sheep anti-influenza virus HA serum served as positive controls in the corresponding HI assay. Samples were visually read; in agglutinated samples, RBCs formed a pattern, while non-agglutinated RBCs formed teardrop-shaped patterns in the center of the wells. The HI titer was defined as the reciprocal of the last serum dilution that inhibited agglutination.

[0367] Enzyme-linked lectin assay (ELLA) To assess the immunogenicity of the mRNA composition against the NA antigen, an enzyme-linked lectin assay (ELLA) was performed. This assay allows for the measurement of antibodies that inhibit the enzymatic activity of NA. In short, a 96-well plate was coated with carbohydrate-containing fetoglobulin, which was then exposed to NA by a single-cycle pseudovirus (PV) carrying NA, used as a substitute virus. The NA enzyme cleaves terminal sialic acid residues from the fetoglobulin, exposing galactose, which is then conjugated to a peanut lectin (PNA-HRPO) conjugated with horseradish peroxidase. This reagent then forms the basis for a colorimetric readout of NA activity. This activity can be inhibited by antibodies present in the serum of vaccinated mice. To measure ELLA titers, each serum sample was heat-treated and then serially diluted in PBS-BSA. 50 µl of each dilution was added to duplicate wells of the fetoglobulin-coated plate. An equal volume (50 µl) of the selected pseudovirus dilution was added to all wells containing serum and at least four wells containing diluent but without serum, serving as a positive (pseudovirus-only) control. Incubate the plate at 37°C for 16–18 h. Wash the plate and add PNA-HRPO to all wells as described for virus titration. After 2 h of incubation, wash the plate and add o-phenylenediamine dihydrochloride (OPD) (Sigma, St. Louis, MO, USA) substrate. Terminate the reaction by adding hydrochloric acid and read the absorbance at 490 nm. ELLA titer is defined as the reciprocal of the last dilution that results in at least 50% inhibition.

[0368] Cellular immune response was detected by ICS. Two weeks after the second vaccination, T-cell immune responses were analyzed by intracellular cytokine staining (ICS) in isolated spleen cells stimulated with a custom 15-mer overlapping peptide library spanning full-length HA and NA proteins. Thawed spleen cells were seeded in 96-well plates. Cells were stimulated with a HA or NA peptide library at 0.5 µg / ml / peptide in the presence of anti-CD28 antibody. After incubation at 37°C for 1 h, GolgiPlug containing brefeldin A was added, and cells were incubated at 37°C for another 5–6 h. Subsequently, spleen cells were washed twice in PBS and incubated at 4°C for 30 min with a live / dead fixable aqua dye. In the presence of Fcγ blocking reagent, cells were stained with antibodies against CD90.2(Thy1.2)-FITC, CD8-APC-Cy7, and CD4-BD HorizonV450 in PBS / 0.5% BSA at 4°C for 30 min. Cells were then washed and fixed. Finally, cells were incubated at 4°C with anti-IFNγ-APC and anti-TNF-PE for 30 min. For FACS analysis, spleen cells were resuspended in PBS / 0.5% BSA / 2 mM EDTA. Cells were collected using a ZE5 flow cytometer (Biorad), and data were analyzed using software.

[0369] result - Clinical observation and local effects Mice were intramuscularly immunized on days 0 and 21 with 0.5 µg of the indicated formulation (A (PSN), B (PSN / TSN), and C (PSN / TSG)) stored at -70°C (T0) or at 25°C for 21 days (21d25°C) (n=14). Control animals (n=5) received the PSN matrix (lacking LNP mRNA) intramuscularly on days 0 and 21. Muscle swelling was scored after each immunization and compared to uninjected muscle: none (score=0), mild (score=1), moderate (score=2), and significant (score=3).

[0370] Mild muscle swelling was observed at most after the first and second administration of the formulation at T0 and 21d at 25°C (see Figure 16), which was comparable between groups.

[0371] - Induction of serum cytokines and chemokines Mice were intramuscularly immunized on days 0 and 21 with 0.5 µg of the indicated formulations (A (PSN), B (PSN / TSN), and C (PSN / TSG)) stored at -70°C (T0) or at 25°C for 21 days (21d25°C) (n=14). Control animals (n=5) received the PSN matrix (lacking LNP mRNA) intramuscularly on days 0 and 21. Levels of IFNγ, CCL2, and CXCL10 were determined in serum samples collected 18 h after the first immunization using a cell counting bead array (CBA).

[0372] Early innate immune responses were assessed by determining serum levels of pro-inflammatory cytokines and chemokines 18 h post-primary immunization. As shown in Figure 17, administration of formulations A, B, and C induced responses in IFNγ, CCL2, and CXCL10. Overall, formulation “PSN / TSN” (B) showed the lowest cytokine induction.

[0373] - Induction of humoral and cellular responses On day 0 and day 21, mice were intramuscularly immunized with 0.5 µg of the indicated formulation (A (PSN), B (PSN / TSN), and C (PSN / TSG)) stored at -70°C (T0) or at 25°C for 21 days (21d25°C) (n=14).

[0374] The induction of functional antibodies was analyzed in serum samples collected three weeks after the first immunization and two weeks after the second immunization. The HI assay was used for anti-HA responses, while the ELLA assay was used for anti-NA responses. The results are presented in Figure 18.

[0375] - Induction of humoral and cellular responses On day 0 and day 21, mice were intramuscularly immunized with 0.5 µg of the indicated formulation (A (PSN), B (PSN / TSN), and C (PSN / TSG)) stored at -70°C (T0) or at 25°C for 21 days (21d25°C) (n=14).

[0376] Two weeks after the second vaccination, T-cell immune responses (CD4 and CD8) were analyzed by ICS in isolated spleen cells stimulated with a custom 15-mer overlapping peptide library spanning full-length HA and NA proteins. The results are presented in Figure 19.

Claims

1. An aqueous composition comprising an RNA molecule, a delivery system, Tris, a sugar, and a phosphate anion.

2. The aqueous composition according to claim 1, wherein Tris is present at a concentration between 5 mM and 50 mM, suitably between 7.5 mM and 30 mM, for example at a concentration of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mM, suitably at a concentration of about 15 mM.

3. The aqueous composition according to claim 1 or 2, wherein the sugar is a disaccharide, suitably selected from sucrose, trehalose, and combinations thereof.

4. The aqueous composition according to claim 3, wherein the disaccharide is sucrose, and wherein the sucrose is present at a concentration between 50 mM and 700 mM, suitably between 100 mM and 600 mM, for example at a concentration of about 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM, suitably at a concentration of about 300 mM.

5. The aqueous composition according to claim 3, wherein the disaccharide is a combination of sucrose and trehalose, and wherein the total concentration of sucrose and trehalose is between 50 mM and 700 mM, suitably between 100 mM and 600 mM, for example 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM, and wherein, for example, sucrose and trehalose are both present at a concentration of about 150 mM, or sucrose is present at a concentration of 50 mM and trehalose is present at a concentration of about 550 mM.

6. The aqueous composition according to any one of claims 1 to 5, wherein the phosphate anion is present at a concentration of 0.5 mM to 15 mM, suitably 1 mM to 10 mM, more preferably 1 mM to 5 mM, for example at a concentration of about 1, 2, 3, 4 or 5 mM, suitably at a concentration of 3 mM.

7. The aqueous composition according to any one of claims 1 to 6, further comprising sodium chloride, suitably present at a concentration between 5 mM and 50 mM, suitably between 10 mM and 40 mM, more suitably between 15 mM and 30 mM, for example at a concentration of about 15, 20, 21, 22, 22.5, 23, 24, 25 or 30 mM, suitably at a concentration of about 22.5 mM.

8. The aqueous composition according to any one of claims 1 to 7, wherein the pH of the aqueous composition is between 6.5 and 8.5, for example, about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, suitably about 7.4 or about 8.

0.

9. The aqueous composition according to any one of claims 1 to 8, further comprising glycerol suitably present at a concentration of 10 mM to 200 mM, for example about 25, 50, 75, 100 or 150 mM, more preferably about 25 mM or about 100 mM.

10. The aqueous composition according to claim 9, wherein the total concentration of sugar and glycerol is between 50 mM and 700 mM, suitably between 100 mM and 600 mM, for example about 150, 200, 250, 300, 350, 400, 450, 500 or 550 mM, and wherein, for example, the sugar is sucrose and is present at a concentration of about 150 mM, and the glycerol is present at a concentration of about 100 mM.

11. The aqueous composition according to any one of claims 1 to 10, wherein a. Tris exists at concentrations ranging from 5 mM to 50 mM, preferably from 7.5 mM to 30 mM. b. The sugar, preferably sucrose, is present at a concentration ranging from 50 mM to 700 mM, preferably from 100 mM to 600 mM. c. The phosphate anion is present at a concentration ranging from 0.5 mM to 15 mM, suitably from 1 mM to 5 mM. d. Sodium chloride is present at a concentration ranging from 5 mM to 50 mM, preferably from 15 mM to 30 mM, and The pH of the aqueous composition is between 6 and 9, preferably between 6.5 and 8.

5.

12. The aqueous composition according to claim 11, wherein glycerol is present at a concentration ranging from 10 mM to 200 mM, suitably from 25 mM to 175 mM.

13. The aqueous composition according to any one of claims 1 to 12, wherein a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. c. Phosphate is present at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of the aqueous composition is approximately 7.

4.

14. The aqueous composition according to any one of claims 1 to 12, wherein a. Tris exists at a concentration of approximately 30 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. c. The phosphate anion is present at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of the aqueous composition is approximately 7.

4.

15. The aqueous composition according to any one of claims 1 to 12, wherein a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 550 mM. c. The phosphate anion is present at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of the aqueous composition is approximately 7.

4.

16. The aqueous composition according to any one of claims 1 to 12, wherein a. Tris exists at a concentration of approximately 15 mM. b. The sugar is sucrose and exists at a concentration of approximately 300 mM. c. The phosphate anion is present at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of the aqueous composition is about 8.

0.

17. The aqueous composition according to any one of claims 1 to 12, wherein a. Tris exists at a concentration of approximately 30 mM. b. The sugar is sucrose and exists at a concentration of approximately 150 mM. c. The phosphate anion is present at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM. e. Glycerin exists at a concentration of approximately 100 mM, and The pH of the aqueous composition is approximately 7.

4.

18. The aqueous composition according to any one of claims 1 to 12, wherein a. Tris exists at a concentration of approximately 10 mM. b. The sugar is sucrose and exists at a concentration of about 225 mM. c. The phosphate anion is present at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM. e. Glycerin is present at a concentration of approximately 25 mM, and The pH of the aqueous composition is approximately 7.

4.

19. The aqueous composition according to any one of claims 1 to 20, wherein a. Tris exists at a concentration of approximately 15 mM. b. The sugar is a combination of sucrose at a concentration of about 150 mM and trehalose at a concentration of about 150 mM. c. The phosphate anion is present at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of the aqueous composition is approximately 7.

4.

20. The aqueous composition according to any one of claims 1 to 12, wherein a. Tris exists at a concentration of approximately 15 mM. b. The sugar is a combination of sucrose at a concentration of about 50 mM and trehalose at a concentration of about 500 mM. c. The phosphate anion is present at a concentration of approximately 3 mM. d. Sodium chloride was present at a concentration of approximately 22.5 mM, and The pH of the aqueous composition is approximately 7.

4.

21. The aqueous composition according to any one of claims 1 to 12, wherein the RNA molecule is an mRNA encoding a protein of interest, suitably selected from non-replicating mRNAs and self-replicating mRNAs.

22. The aqueous composition of claim 21, wherein the protein of interest is an antigen, suitably selected from respiratory virus antigens such as influenza HA antigen, influenza NA antigen, SARS-CoV-2 S antigen, RSV F antigen, HMPV F antigen, and PIV F antigen.

23. The aqueous composition according to any one of claims 1 to 22, wherein the delivery system is lipid nanoparticles (LNPs) suitably comprising (i) ionizable amino lipids, (ii) neutral lipids, suitably DSPC, (iii) sterols, suitably cholesterol; and (iv) PEGylated lipids.

24. The aqueous composition according to any one of claims 1 to 23, for use in therapy, suitably as a vaccine.

25. A method for preparing the aqueous composition according to claims 1 to 23, comprising: a) Provide an initial composition comprising an RNA molecule, preferably mRNA, and a delivery system, preferably LNP. b) Transfer the initial composition to a first buffer system to obtain an intermediate composition. c) Dilute the intermediate composition with a dilution buffer to obtain an aqueous composition as defined in claims 1 to 23.

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